Plant viral expression vectors and use of same for generating genotypic variations in plant genomes

ABSTRACT

A method of generating genotypic variation in a genome of a plant is disclosed. The method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to plantviral expression vectors and, more particularly, but not exclusively, tothe use of same for generating genotypic variations in plant genomes.

Genetic modification and improvement of crop plants as well asprotection of new varieties is fundamental for modern agriculture.During the past several years an enormous amount of data was obtainedfrom the various large genome-sequencing projects allowing significantprogress in agriculture transgenic technologies. Such technologies,including gene expression, gene modification, site-specific genemutagenesis and gene targeting of plant genome sequences, allowdevelopment of basic plant research models and can be directly used forgenetic improvement and protection of agronomically important plantspecies.

Foreign DNA molecules (e.g. T-DNA) delivered by Agrobacterium areintegrated in the plant's genome into natural double strand breaks(DSBs) which may be generated by rare-cutting restriction enzymes. TheseDSBs are recognized and repaired by plant non-homologous end joining(NHEJ) proteins and results in the frequent integration of the foreignDNA into these random sites [Salomon et al. EMBO J (1998) 17: 6086-6095;Tzfira et al. Plant Physiol (2003) 133: 1011-1023, Tzfira et al. TrendsGenet (2004) 20: 375-383]. The DSBs may also result in enhancedhomologous recombination (HR)-based gene targeting in plant cells[Puchta et al. Proc Natl Acad Sci USA (1996) 93: 5055-5060].

Recent developments in the field of zinc finger nucleases (ZFNs) asnovel tools for genome modifications offer new prospects forsite-specific induction of DSBs in plant genomes and for the developmentof NHEJ-based methods for gene targeting in plant species and plantprotection. ZFNs are synthetic restriction enzymes which can bespecifically designed to bind and cleave virtually any long stretch ofdsDNA sequences (see FIG. 1). ZFNs were shown suitable for site-specificgenomic DSB induction in plant species using non-viral vectors [Lloyd etal. Proc. Natl. Acad. Sci. U.S. A. (2005) 102: 2232-2237; Tovkach et al.The Plant Journal (2009) 57, 747-757]. Similar effects were shown onhuman [Moehle et al. Proc Natl Acad Sci USA (2007) 104: 3055-3060] andinsect genomes [Beumer et al. Genetics (2006) 172: 2391-2403].

The use of plant viruses as vehicles to introduce and express nonviralgenes in plants is well documented [e.g. Donson et al., Proc Natl AcadSci USA. (1991) 88: 7204-8; Chapman et al. Plant J. (1992) 2: 549-57;Dolja et al., Virology (1998) 252: 269-74]. Infection of plants withmodified viruses is simpler and quicker than the regeneration of stablytransformed plants (as discussed above) since plant viruses are oftensmall in size (between 3000 and 10,000 nucleotides), are easy tomanipulate, have the inherent ability to enter the plant cell, lead tothe immediate expression of the heterologous gene and will multiply toproduce a high copy number of the gene of interest. Viral vectors havebeen engineered for delivery of genetic material and expression ofrecombinant proteins in plants [e.g., Pogue, Annu. Rev. Phytopathol.(2002) 40: 45-74; Gleba, et al., Curr. Opin. Plant Biol. (2004) 7:182-188; Dolja et al., Proc. Natl. Acad. Sci. USA (1992) 89:10208-10212; U.S. Pat. No. 5,316,931 and U.S. Pat. No. 5,811,653 for RNAvirus vectors]. Viral expression systems are considered transientexpression systems as the viral vectors are not integrated into thegenome of the host, however, depending on which virus is used, virusmultiplication and gene expression can persist for long periods (up toseveral weeks or months).

To date the use of viral vectors for introducing DSBs in plant genomeswas not demonstrated or suggested.

RELATED ART

U.S. Pat. No. 7,229,829 discloses TRV vectors (TRV-RNA1 and TRV-RNA2)carrying heterologous nucleic acid sequences for delivery into plantsfor transforming plants and plant cells. Specifically, U.S. Pat. No.7,229,829 teaches vectors for virus induced gene silencing (VIGS)including vectors designed for suppression of host plant gene expression(e.g. antisense transcripts for knocking out expression of genes withoutthe need to genetically transform the plant) or vectors designed forexpression of heterologous nucleic acids (e.g. nucleic acids mediatinggene silencing or gene suppression).

U.S. Publication Nos. 20050026157 and 20070134796 discloses compositionsand methods for targeted cleavage of cellular chromatin and for targetedalterations (e.g. insertions) of cellular nucleotide sequences. Totarget specific genomic sites, fusion proteins are constructed whichcomprise a zinc finger domain and a cleavage domain [i.e. zinc fingerproteins (ZFPs)]. Moreover, U.S. Publication No. 20070134796 teachesvectors (e.g. bacterial vectors such as plasmid vectors and viralvectors such as adenoviral and retroviral vectors) comprising the ZFPs.

PCT Publication No. WO07139982 discloses methods and compositions forinactivating human genes (e.g. CCR5 gene) using zinc finger nucleases(ZFNs). The ZFNs comprise a zinc finger protein (may include 1, 2, 3, 4,5, 6 or more zinc fingers) and a cleavage domain or cleavage half-domain(i.e., a nuclease domain). Furthermore, PCT Publication No. WO07139982teaches vectors comprising ZFNs and/or a donor sequence for targetedintegration into a target gene.

U.S. Pat. Nos. 7,309,605 and 6,610,545 disclose nucleotide sequencesencoding the enzyme I-SceI (a double-stranded endonuclease that cleavesDNA within its recognition site). These sequences can be incorporated incloning and expression vectors (such as plasmid, bacteriophage or cosmidvectors) and may be used to transform cell lines and transgenicorganisms (e.g. mammals, plants). The vectors disclosed are useful ingene mapping, in site directed genetic recombination and in in-vivosite-directed insertion of genes.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of generating genotypic variation in a genomeof a plant, the method comprising introducing into the plant at leastone viral expression vector encoding at least one chimeric nucleasewhich comprises a DNA binding domain, a nuclease and a localizationsignal to a DNA-containing organelle, wherein the DNA binding domainmediates specific targeting of the nuclease to the genome of the plant,thereby generating genotypic variation in the genome of the plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a plant infection by a pathogen,the method comprising introducing into the plant at least one viralexpression vector encoding at least one chimeric nuclease whichcomprises a DNA binding domain and a nuclease, wherein the DNA bindingdomain mediates targeting of the nuclease to the genome of the pathogen,thereby preventing or treating a plant infection by a pathogen.

According to an aspect of some embodiments of the present inventionthere is provided a method of tagging a genome of a plant, the methodcomprising introducing into the plant at least one viral expressionvector encoding at least one chimeric nuclease which comprises a DNAbinding domain, a nuclease and a localization signal to a DNA-containingorganelle, wherein the DNA binding domain mediates specific targeting ofthe nuclease to the genome of the plant, thereby tagging the genome ofthe plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating male sterility in a plant, themethod comprising upregulating in the plant a structural or functionalgene of a mitochondria or chloroplast associated with male sterility byintroducing into the plant at least one viral expression vector encodingat least one chimeric nuclease which comprises a DNA binding domain, anuclease and a mitochondria or chloroplast localization signal and anucleic acid expression construct which comprises at least oneheterologous nucleic acid sequence which can upregulate the structuralor functional gene of a mitochondria or chloroplast when targeted intothe genome of the mitochondria or chloroplast, wherein the DNA bindingdomain mediates targeting of the heterologous nucleic acid sequence tothe genome of the mitochondria or chloroplast, thereby generating malesterility in the plant.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a herbicide resistant plant,the method comprising introducing into the plant at least one viralexpression vector encoding at least one chimeric nuclease whichcomprises a DNA binding domain, a nuclease and a chloroplastlocalization signal, wherein the DNA binding domain mediates targetingof the nuclease to a gene conferring sensitivity to herbicides, therebygenerating the herbicide resistant plant.

According to an aspect of some embodiments of the present inventionthere is provided a plant viral expression vector comprising a nucleicacid sequence encoding at least one chimeric nuclease which comprises aDNA binding domain, a nuclease and a localization signal to aDNA-containing organelle.

According to an aspect of some embodiments of the present inventionthere is provided a pTRV based expression vector comprising a nucleicacid sequence encoding at least two heterologous polypeptide sequences.

According to an aspect of some embodiments of the present inventionthere is provided a plant cell comprising at least one chimericnuclease, wherein the chimeric nuclease comprises a DNA binding domain,a nuclease and a localization signal to a DNA-containing organelle, andwherein the chimeric nuclease induces cleavage of a target sequence.

According to an aspect of some embodiments of the present inventionthere is provided a transgenic plant comprising the plant viralexpression vector of claim 6 or 7.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating a transgenic plant, the methodcomprising: introducing into one or more cells of the plant at least oneviral expression vector encoding at least one chimeric nuclease whichcomprises a DNA binding domain, a nuclease and a localization signal toa DNA-containing organelle.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence as set forth in SEQ ID NOs: 31, 32, 33, 34, 70, 72, 74, 76, 84,86 or 88.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polypeptide comprising an amino acidsequence as set forth in SEQ ID NOs: 35, 36, 37, 38, 71, 73, 75, 77, 85,87 or 89.

According to some embodiments of the invention, generating genotypicvariation is transient.

According to some embodiments of the invention, the genotypic variationcomprises a nucleotide insertion, a nucleotide deletion or a combinationof same.

According to some embodiments of the invention, the tagging comprises anucleotide insertion, a nucleotide deletion or a combination of same.

According to some embodiments of the invention, the viral expressionvector comprises a Tobacco Rattle Virus (TRV) expression vector.

According to some embodiments of the invention, the TRV expressionvector comprises a pTRV2 based expression vector.

According to some embodiments of the invention, at least one viralexpression vector encodes for two chimeric nucleases.

According to some embodiments of the invention, the at least one viralexpression vector comprises two viral expression vectors.

According to some embodiments of the invention, the two viral expressionvectors are introduced into the plant concomitantly.

According to some embodiments of the invention, introducing into theplant is effected by an Agrobacterium.

According to some embodiments of the invention, the Agrobacterium iseffected by injection.

According to some embodiments of the invention, introducing theAgrobacterium is effected by leaf infiltration.

According to some embodiments of the invention, introducing into theplant is effected by virion infection.

According to some embodiments of the invention, the at least onechimeric nuclease comprises two chimeric nucleases.

According to some embodiments of the invention, the plant viralexpression vector or transgenic plant further comprises a second nucleicacid sequence encoding a heterologous polypeptide.

According to some embodiments of the invention, the plant viralexpression vector or transgenic plant comprises a pTRV backbone.

According to some embodiments of the invention, the pTRV is a pTRV1(GeneBank Accession No: AF406990).

According to some embodiments of the invention, the pTRV is a pTRV2(GeneBank Accession No: AF406991).

According to some embodiments of the invention, the nucleic acidsequence is devoid of a 2b sequence (SEQ ID NO: 43).

According to some embodiments of the invention, the nucleic acidsequence comprises a Ω enhancer (SEQ ID NO: 44).

According to some embodiments of the invention, the nucleic acidsequence comprises two separate sub genomic promoters (sgPs) forregulating transcription of the at least two heterologous polypeptides.

According to some embodiments of the invention, the at least twoheterologous polypeptide sequences are separated by nucleic acidsequence encoding a cleavage domain.

According to some embodiments of the invention, the cleavage domaincomprises a T2A-like protein sequence (SEQ ID NO: 40).

According to some embodiments of the invention, the nucleic acidsequence of the at least two heterologous polypeptide sequences is asset forth in SEQ ID NOs: 84, 86 or 88.

According to some embodiments of the invention, the amino acid sequenceof at least two heterologous polypeptide sequences are as set forth inSEQ ID NOs: 85, 87 or 89.

According to some embodiments of the invention, the at least twoheterologous polypeptide sequences encode for a plant gene.

According to some embodiments of the invention, the at least twoheterologous polypeptide sequences comprise chimeric proteins, whereineach of the chimeric proteins comprise a DNA binding domain, a nucleaseand a localization signal to a DNA-containing organelle.

According to some embodiments of the invention, the localization signalcomprises a ribulose-1,5-bisphospate carboxylase small subunit (RSSU)sequence (SEQ ID NO: 138).

According to some embodiments of the invention, the localization signalcomprises an ATPase beta subunit (ATP-β) sequence (SEQ ID NO: 139).

According to some embodiments of the invention, the DNA binding domainbinds a 9 nucleotide sequence.

According to some embodiments of the invention, the DNA binding domaincomprises at least one zinc finger domain.

According to some embodiments of the invention, the zinc finger domaincomprises three zinc finger domains.

According to some embodiments of the invention, the nuclease comprises acleavage domain of a type II restriction endonuclease.

According to some embodiments of the invention, the type II restrictionendonuclease is a FokI restriction endonuclease.

According to some embodiments of the invention, the plant comprises aPetunia hybrida.

According to some embodiments of the invention, the plant comprises aNicotiana tabacum.

According to some embodiments of the invention, the plant in selectedfrom the group consisting of an Arabidopsis thaliana, an Artemisia sp.,a Artemisia annua, a Beta vulgaris, a Solanum tuberosum, a Solanumpimpinellifolium, a Solanum lycopersicum, a Solanum melongena, aSpinacia oleracea, a Pisum sativum, a Capsicum annuum, a Cucumissativus, a Nicotiana benthamiana, a Nicotiana tabacum, a Zea mays, aBrassica napus, a Gossypium hirsutum cv. Siv'on, a Oryza sativa and aOryza glaberrima.

According to some embodiments of the invention, the cell is a meristemcell.

According to some embodiments of the invention, the DNA-containingorganelle is selected from the group consisting of a nucleus, achloroplast and a mitochondria.

According to some embodiments of the invention, the specific targetingof the nuclease to the genome of the Petunia hybrida is to a phytoenedesaturase (PDS) or a flavanone 3 beta-hydroxylase (FHT) of the Petuniahybrida.

According to some embodiments of the invention, the mitochondrialocalization signal comprises an ATPase beta subunit (ATP-β) (SEQ ID NO:139).

According to some embodiments of the invention, the chloroplastlocalization signal comprises a ribulose-1,5-bisphospate carboxylasesmall subunit (Rssu) (SEQ ID NO: 138).

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a schematic illustration of Zinc-finger nucleases (ZFNs) as atool for the induction of genomic double-strand breaks (DSBs). FIG. 1Adepicts the structure of ZFNs chimeric genes composed of a syntheticDNA-recognition domain consisting of three C2H2 zinc fingers fused to anon-specific DNA restriction enzyme (usually the FokI endonuclease);FIG. 1B depicts custom-made ZFN genes, in which each finger recognizes athree-nucleotide sequence and can potentially be designed to recognizeany combination of nine nucleotides (exemplified here by a GGGGAAGAAtarget sequence, SEQ ID NO: 42). Since FokI functions as a dimer, twosets of ZFNs are used to bind the target DNA, which results in a uniquecombination of 18 nucleotides; FIG. 1C depicts binding of the ZFNs tothe target DNA; FIG. 1D depicts digestion of the DNA by FokIendonuclease domain and creation of a double-strand break (DSB). FIG. 1Edepicts repair of the DSBs by non-homologous end-joining (NHEJ) proteinsthat lead to deletion or insertion mutations at the repair site.

FIG. 2A shows sequence alignment between the sequence of Thosea asignavirus for the self cleaving peptide (Tav-T2A, SEQ ID NO: 51) and themodified sequence according to the codon usage of Petunia (pTRV-T2A, SEQID NO: 52).

FIG. 2B shows P1-25 Petunia RB random DNA fragment (SEQ ID NO: 8).

FIG. 3 shows P1-36 Petunia RB random DNA fragment (SEQ ID NO: 9).

FIG. 4 is a schematic illustration of a ZFN structure constructedaccording to the present teachings. The ZFN construct comprises anuclear localization signal (NLS), a Zink Finger DNA binding domain anda DNA nuclease domain from FokI.

FIGS. 5A-D shows the sequences of NLS-P1-25-ZFN1, NLS-P1-25-ZFN2,NLS-P1-36-ZFN1 and NLS-P1-36-ZFN2. The sequence is of the full chimera:NLS, ZFN and FokI (d-domain). Sequences of nuclear localization signal(NLS) are depicted in lower case; nuclease (FokI-domain d) first codonand the termination codon are depicted in bold. FIG. 5A shows thesequence of NLS-P1-25-ZFN1 (SEQ ID NO: 31); FIG. 5B shows the sequenceof NLS-P1-25-ZFN2 (SEQ ID NO: 32); FIG. 5C shows the sequence ofNLS-P1-36-ZFN1 (SEQ ID NO: 33); and FIG. 5D shows the sequence ofNLS-P1-36-ZFN2 (SEQ ID NO: 34).

FIG. 5E is a scheme showing the original pET28 vector (SEQ ID NO: 39,commercial available from Novagen) and the modified pET28 vector (SEQ IDNO: 49), pET28c.SX, comprising a modification of MCS. To constructpET28c-SX, nucleotides 179 to 158 (indicated by bold) were deleted fromMSC of pET28c by digestion with SalI and XhoI.

FIGS. 6A-D are schematic maps of pTRV2 (GenBank accession No. AF406991)and its modifications. FIG. 6A is a schematic illustration of thecomplete pTRV2 expression vector; FIG. 6B depicts the removal of the 2b5′ CDS fragment; FIG. 6C depicts the addition of 5′UTR of TMV (a); andFIG. 6D depicts the addition of sgP-CP from PEBV.

FIG. 6E is schematic maps of part of pTRV1 showing RNA1 (GenBankaccession No. AF406990).

FIGS. 7A-E are pictures illustrating the expression of GUS in meristemsof petunia plants inoculated with pTRV2-GUS and pTRV2-Δ2b-GUS. FIGS.7A-B depict meristems from petunia plants 7 days after stem inoculationwith the vectors; and FIGS. 7C-D depict meristems from petunia plants 37days after stem inoculation with the vectors. Plants inoculated withpTRV2-GUS are shown in FIGS. 7A and 7C. Plants inoculated withpTRV2-Δ2b-GUS are shown in FIGS. 7B and 7D. FIG. 7E shows GUS stainingin petunia plants propagated in vitro 6 month following inoculation withpTRV2-Δ2b-GUS.

FIGS. 8A-G are pictures illustrating the expression of marker genes invarious plants following inoculation with pTRV2 based vectors. FIG. 8Adepicts GUS staining (24 days post inoculation) of pepper plants(Capsicum annuum, Endra-1750) inoculated with pTRV2-Δ2b-GUS; FIG. 8Bdepicts GFP staining (41 days post inoculation) of pepper plants(Capsicum annuum, Endra-1750) inoculated with pTRV2-Δ2b-GFP; FIG. 8Cdepicts GUS staining (13 and 30 days post inoculation) of Arabidopsisplants inoculated with pTRV2-GUS; FIG. 8D depicts GUS staining (14 dayspost inoculation) of tomato plants (Solanum pimpinellifolium La121)inoculated with pTRV2-Δ2b-ΩGus; FIG. 8E depicts GFP staining (31 dayspost inoculation) of Nicotiana benthamiana plants inoculated withpTRV2-Δ2b-GFP, FIG. 8F depicts GUS staining (44 days post inoculation)of Nicotiana benthamiana plants inoculated with pTRV2-35SΩGUS; and FIG.8G depicts pigmentation of Nicotiana benthamiana inoculated withpTRV2-Δ2b-PAP (76 days post inoculation).

FIGS. 9A-B are pictures illustrating GUS staining 51 days postinoculation of petunia plants with pTRV2-Δ2b-ΩGUS (FIG. 9A) as comparedto pTRV2-Δ2b-GUS (FIG. 9B). Of note, arrows point to meristematicregions.

FIGS. 10A-C are pictures illustrating co-expression of two genes inmeristems of N. benthamiana plants. FIG. 10A depicts plants inoculatedwith pTRV2-Δ2b-sgP-GFP. GFP staining was evaluated 17 days postinoculation with pTRV2-Δ2b-sgP-GFP; FIGS. 10B-C depict plantsco-inoculated with pTRV2-Δ2b-GUS and pTRV2-Δ2b-GFP. GFP staining wasevaluated 17 days post inoculation (FIG. 10B) followed by GUS stainingon the same tissue sample (FIG. 10C).

FIGS. 11A-F are pictures illustrating virus-mediated gene expression(DsRed) in cells of different plants. Plants were inoculated with pTRV1and pTRV2-Δ2b-sgP-DsRed and fluorescence was evaluated using confocallaser scanning microscopy. Upper panel (FIGS. 11A, C, E) shows cells'chlorophyll autofluorescence and lower panel (FIGS. 11B, D, F) showsDsRed fluorescence in the same cells. Autofluorescence was evaluated atexcitation (Ex) 488 nm and emission (Em) at more than 650 nm, DsRed wasevaluated at ex 545 nm and em between 585-615 nm.

FIGS. 12A-H are pictures illustrating long term gene expression (DsRed)in different plants following inoculation with pTRV1 andpTRV2-Δ2b-sgP-DsRed. Images were obtained using fluorescentstereomicroscope. DsRed fluorescence (FIGS. 12A, C, D, G) was visible indifferent parts of plants, including roots (FIGS. 12D, F). Lower panel(FIGS. 12B, E, F, H) shows (the same) images taken under bright field.Of note, N. Benthamiana was innoculated with TRV virions and N. tabaccumand Petunia hybrida were innoculated via agro-infiltration.

FIGS. 13A-K are pictures illustrating the applicability of the TRV2vector for expression of foreign genes in various plants. Expression ofthe marker genes GUS, GFP and DsRed was demonstrated in various plantsbelonging to different families following inoculation with TRV vectors:Beta vulgaris (FIG. 13E) were inoculated with pTRV-Δ2b-GUS (sample GUSstained 20 days post inoculation); Solanum melongena (FIGS. 13F-G) wasinoculated with pTRV-Δ2b-sgP-Rssu-EGFP (evaluated 5 days postinoculation); Cucumis sativus (FIGS. 13A-B), Gossypium hirsutum cv.Siv'on (FIGS. 13H-I) and Brassica napus (FIGS. 13J-K) were inoculatedwith pTRV-Δ2b-sgP-DsRed (evaluated 5, 7 and 16 days post inoculation,respectively). Bright field images were also demonstrated. Spinaciaoleracea (FIGS. 13C-D) was inoculated with pTRV-Δ2b-GUS (FIG. 13C,sample GUS stained 20 days post inoculation) and pTRV-Δ2b-sgP-DsRed(FIG. 13D, evaluated 12 days post inoculation). All images were takenusing fluorescent stereomicroscope.

FIGS. 14A-C are pictures illustrating expression of DsRed2 in Zea maysVar. Royalty coleoptile by TRV viral vectors. Seeds were inoculated withsap containing diluted virions, extracted from Petunia agroinfiltratedwith pTRV1 and pTRV2-Δ2b-sgP-DsRed. DsRed was evaluated at 16 days postinoculation (dpi). Visible (FIG. 14A), DsRed (FIG. 14B) and Merged (FIG.14C).

FIGS. 15A-G are pictures illustrating chloroplast-targeted expression ofEGFP in petunia and tobacco following infection withpTRV2-sgP-Rssu-EGFP. N. Tabacuum cv Samsung and Petunia hybrida CV. RBwere inoculated with pTRV2-sgP-Rssu-EGFP and the expression of EGFP inchloroplasts was assayed approximately 9 days post inoculation.Autofluorescence (FIGS. 15A, E) of chlorophyll: was evaluated atexcitation (ex) 488 nm and emission (em) was evaluated at more than 650nm. The EGFP (FIGS. 15B, F) was detected by ex at 488 and em between505-530 nm. Merged signal (FIGS. 15D and G, overlay of pink and greenyielding yellow). Inset (FIG. 15C) shows tissue expressing EGFPvisualized by fluorescent stereomicroscope.

FIGS. 16A-K are pictures illustrating mitochondrial-targeted expressionof EGFP in petunia following infection with pTRV2-sgP-ATPβ-EGFP. Petuniahybrida CV. RB were inoculated with pTRV2-sgP-ATPβ-EGFP and expressionof EGFP in mitochondria was assayed approximately 5 days postinoculation. Autofluorescence (FIG. 16A) was evaluated at ex 488 and emat more than 650 nm. EGFP (FIG. 16B) was evaluated at ex. 488 and embetween 505-530 nm. Protoplast (FIGS. 16E-K) were prepared from PetuniaRB expressing mitochondrial targeted EGFP, stained with MitoTracker andevaluated at ex 545 nm and em between 585-615 nm. Inset (FIG. 16G) showstissue expressing EGFP visualized by fluorescent stereomicroscope.

FIGS. 17A-D are pictures illustrating co-expression of marker genes indifferent cellular compartments. DsRed was expressed in the cytosol andGFP in the chloroplasts of N. tabacum cv. Xanthi leaf cells. Plants wereco-infected with pTRV1 and pTRV2-Δ2b-sgP-DsRed andpTRV2-Δ2b-sgP-Rssu-EGFP. Autofluorescence (FIG. 17A) was evaluated usingex 488 nm and em of more than 650 nm, GFP (FIG. 17B) was evaluated usingex 488 nm and em between 505-530 nm, DsRed (FIG. 17C) was evaluatedusing ex 545 nm and em between 585-615 nm, Merge (FIG. 17D) depicts amerge of all three filters (merged signals).

FIGS. 18A-L are pictures illustrating co-expression of DsRed and EGFP indifferent plants using pTRV2 constructed with the two reporter genes intandem separated by T2A. Plants (Petunia hybrida, N. tobaccum and N.benthamiana) were inoculated with pTRV1 andpTRV2-Δ2b-sgP-DsRed-T2A-NLS-EGFP. Fluorescence was evaluated usingconfocal laser scanning microscopy. Cells' chlorophyll autofluorescence(FIGS. 18A, E, I), EGFP (FIGS. 18B, F, J) and DsRed (FIGS. 18C, G, K)and merged signal (FIGS. 18D, H, L) are shown. Autofluorescence wasevaluated at ex 488 nm and em at more than 650 nm, EGFP was evaluated atex 488 nm and em between 505-530 nm, DsRed2 was evaluated at ex 545 nmand em between 585-615 nm.

FIGS. 19A-J are pictures illustrating co-expression of DsRed and GFP indifferent plants using pTRV2 constructed with the two reporter genes intandem driven by separate double subgenomic promoters. Plants (N.tobaccum and N. benthamiana) were inoculated with pTRV1 andpTRV2-Δ2b-sgP-GFP-sgP-DsRed. Fluorescence was evaluated using confocallaser scanning microscopy. The cells' chlorophyll autofluorescence(FIGS. 19A, E), GFP (FIGS. 19B, F) and DsRed (FIGS. 19C, G) are shown.FIG. 19D depicts an image in bright field (for N. tobaccum) and FIG. 19Hdepicts an image of merged signal (for N. benthamiana). FIGS. 19I-J areimages, taken by stereomicroscope, of inoculated N. benthamiana tissues(3 dpi). Autofluorescence ex was evaluated at 488 nm and em at more than650 nm, GFP was evaluated at ex 488 nm and em between 505-530 nm, DsRed2was evaluated at ex 545 nm and em between 585-615 nm.

FIG. 20 is a picture illustrating digestion of PCR fragments carryingartificial target sites P1-25-1, P1-25-2, P1-36-1 and P1-36-2 (P25-TS1,P25-TS2, P36-TS1, P36-TS2 respectively) by specific ZFNs. PCR fragments(ca. 900 bp) carrying palindrome-like target sequences were incubatedwith 25-ZFN-1,25-ZFN-2 or 36-ZFN-1,36-ZFN-2 and the digestion productswere separated by agarose gel. Of note, the TS (target site) ispalindrome-like.

FIG. 21 is a picture illustrating digestion of a NcoI/BamHI (740 bp)fragment from pBS-PI-36, carrying a target P1-36 sequence, with 36-ZFN1and 36-ZFN2.

FIG. 22 is a picture illustrating digestion of plasmid pBS carryingPI-36 (pBS-PI-36) by a mixture of ZFNs (36-ZFN 1 and 2). Fragment ofexpected size (515 bp) is indicated by arrow.

FIG. 23 is a picture illustrating digestion of a plasmid carrying targetPDS1 or PDS2 palindromic sites. The tested palindromic sites were cutwith specific ZFNs (PDS-ZFN1 and PDS-ZFN2) and AgeI which yielded afragment of approximately 950 bp, as expected. Volumes listed above thecolumns refer to the amount of enzyme containing crude extract used. Ofnote, the TS (target site) is palindrome-like.

FIGS. 24A-B are pictures illustrating the expression of DsRFP (FIG. 24B)and GFP (FIG. 24A) in petunia plants inoculated withpTRV2-Δ2b-sgP-CP-PEBV carrying DsRFP and GFP separated by T2A. Figuresdepict leaf tissues from petunia plants 10 days after stem inoculationwith the vector.

FIGS. 25A-B show the N termini of the mutated uidA gene sequence. FIG.25A depicts the uidA gene sequence containing insert of target sites ofQEQ-ZFN (bold) and spacer with stop codon (red). FIG. 25B depicts howmiss-repair of the double strand breaks formed by the QEQ-ZFN may leadto elimination of the stop codon and reconstruction of the uidA gene.

FIGS. 26A-J are pictures illustrating TRV-based repair of uidA inplanta. Transgenic petunia and tobacco plants carrying mutated uidA wereinoculated in vitro or in vivo with pTRV1 and pTRV2-Δ2b-sgP-QEQ-ZFN. Atdifferent times after agroinfiltration or inoculation with virions, GUSactivity was evaluated in various parts of the plant including intissues that developed after inoculation. FIG. 26A depicts a mutateduidA transgenic Petunia hybrida line 65 evaluated for GUS expression 12days post in-vitro agroinoculation. FIG. 26B depicts a mutated uidAtransgenic Nicotiana tobaccum line 3 evaluated for GUS expression 22days post in-vitro agroinoculation. FIG. 26C depicts a mutated uidAtransgenic Petunia hybrida line I evaluated for GUS expression 11 dayspost in-vitro agroinoculation. FIG. 26D depicts a mutated uidAtransgenic Nicotiana tobaccum line 3 evaluated for GUS expression 50days post inoculation. FIG. 26E depicts a mutated uidA transgenicPetunia hybrida line N evaluated for GUS expression 17 days postin-vitro agroinoculation. FIG. 26F depicts a mutated uidA transgenicPetunia hybrida line I in-vitro inoculation with virions evaluated forGUS expression was carried out 15 days post inoculation. FIG. 26Gdepicts a mutated uidA transgenic Petunia hybrida line I in-vitroagroinoculation with 0.08 OD, evaluated 29 days post inoculation. FIG.26H depicts a mutated uidA transgenic Petunia hybrida line I in-vitroagro inoculation with 0.8 OD, evaluated 29 days post inoculation. FIG.26I depicts a mutated uidA transgenic Nicotiana tobaccum line 11, nottreated with TRV, GUS tested, and FIG. 26J depicts a mutated uidAtransgenic Petunia hybrida line I primordia regeneration evaluated forGUS expression, following in-vitro agroinoculation.

FIG. 27 shows alignment of 20 mutant sequences (SEQ ID NOs: 96-116) inmutated uidA (GUS) identified by DdeI site disruption in the GUS stopcodon of transgenic N. tabacum CV Samsung (N_t) as well as Petuniahybrida (Pet) plants. Of note, the results depicted insertions (1 or 2nucleotides) and deletions (less or equal to 49 nucleotides).Restoration of GUS activity can be ascribed to mutation in Pet30.

FIG. 28A shows the sequence of phytoene desaturase (PDS) exon fromPetunia hybrida RB (GenBank accession no AY593974.1, SEQ ID NO: 131).This sequence was confirmed by resequencing (indicated by upper caseletters). The highlighted sequences are the target sites (PDS-ZFN1target site—SEQ ID NO 140 and PDS-ZFN2 target site—SEQ ID NO: 141) ofthe PDS-ZFNs (SEQ ID NOs: 71 and 73) generated by the present invention(SEQ ID NOs: 70 and 72). SEQ ID NO: 132 depicts a short fragment of asequence of the complementary strand to which PDS-ZFN2 binds.Recognition site for the MfeI is underlined.

FIG. 28B shows the changes in the PDS nucleic acid sequences (SEQ IDNOs: 119-128) in Petunia hybrida plants inoculated with pTRV1 andpTRV2-Δ2b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 vectors. The mutants were comparedto the native PDS sequences in the tested Petunia hybrida plants(PDS-WT, Y10 and G35). TS1 (GGAGATGCA, SEQ ID NO: 135) and TS2(CACTTCAAT, SEQ ID NO: 136) indicate the binding sites for ZFNs in thePDS gene. The MfeI site (CAATTG, SEQ ID NO: 137) served as a selectiontool to isolate ZFNs mediated PDS mutants.

FIG. 29 shows the sequence of flavanone 3 beta-hydroxylase (FHT) exonfrom Petunia hybrida cv. RB (GenBank accession no AF022142.1, SEQ ID NO:133). The sequence was confirmed by resequencing. The highlightedsequences (FHT-ZFN1 target site—SEQ ID NO 142 and FHT-ZFN2 targetsite—SEQ ID NO: 143) were used as the target sites for the FHT-ZFNsgenerated by the present invention (SEQ ID NOs: 74 and 76). Recognitionsite for the EcoNI is underlined. SEQ ID NO: 134 depicts a shortfragment of a sequence of the complementary strand to which FHT-ZFN2binds.

FIG. 30 shows the sequence of pTRV2 containing 2b and PEBV-CP subgenomicpromoters (sgP) region (SEQ ID NO: 79). For comparison, the sequencelacking 40 nucleotides from 2b-sgP (pTRV2-Δ2b-Δ2bsgP-sgP) is shown inthe upper line (SEQ ID NO: 78). Nucleotides) to 72-3′ of CP gene,nucleotides 73 to 237—sgP of 2b, nucleotides 238 to 282—MCS, nucleotides283 to 466-sgP of CP from PEBV. Nucleotides 206 to 237—deletion from the3′ of the 2b sgP.

FIG. 31A shows the sequence of NLS-PDS-ZFN1 (SEQ ID NO 70). The sequenceis of the full chimera: NLS, ZFN and FokI (d-domain). Sequences ofnuclear localization signal (NLS) are depicted in lower case; nuclease(FokI-domain d) first codon and the termination codon are depicted inbold.

FIG. 31B shows the sequence of NLS-PDS-ZFN2 (SEQ ID NO 72). The sequenceis of the full chimera: NLS, ZFN and FokI (d-domain). Sequences ofnuclear localization signal (NLS) are depicted in lower case; nuclease(FokI-domain d) first codon and the termination codon are depicted inbold.

FIG. 32A shows the sequence of NLS-FHT-ZFN1 (SEQ ID NO 74). The sequenceis of the full chimera: NLS, ZFN and FokI (d-domain). Sequences ofnuclear localization signal (NLS) are depicted in lower case; nuclease(FokI-domain d) first codon and the termination codon are depicted inbold.

FIG. 32B shows the sequence of NLS-FHT-ZFN2 (SEQ ID NO 76). The sequenceis of the full chimera: NLS, ZFN and FokI (d-domain). Sequences ofnuclear localization signal (NLS) are depicted in lower case; nuclease(FokI-domain d) first codon and the termination codon are depicted inbold.

FIG. 33 shows a schematic representation of pTRV2-Δ2b-sgP-QEQ-ZFN (SEQID NO: 82).

FIG. 34 shows a schematic representation of part of the pTRV2 vectorcarrying a gene-specific ZFNs (PDS is shown for illustration) fusedthrough T2A sequence, downstream to two subgenomic (2b-sgP andPEBV-CP-sgP) promoters.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to plantviral expression vectors and, more particularly, but not exclusively, tothe use of same for generating genotypic variations in plant genomes.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

While reducing some embodiments of the present invention to practice,the present inventors have devised an effective tool for generatinggenotypic variation in plants using viral vectors encoding chimericpolypeptides designed for generating sequence specific double-strandbreaks in the plant's genome. As noted in the background section, theformation of DSBs can be used for passively (by plants' repair system)or actively (i.e., directed insertion of heterologous nucleic acidsequences) generating genotypic variation. The aforementionedsubstantiates beyond any doubt the value of the present tools ingenerating genomic variations.

As is illustrated in the Examples section which follows, the presentinventors have constructed modified tobacco rattle virus (TRV)expression vectors. These vectors were successfully used for introducingand expressing foreign genes of sizes equivalent to the chimeric genesof the present invention (e.g. GUS) in meristematic tissues of differentplants (e.g. Petunia, N. benthamiana and N. Tobaccum, e.g. FIGS. 8A-G,FIGS. 9A-B and FIGS. 11A-F). The present inventors were successful inexpressing heterologous genes in chloroplast and mitochondrial plastids(FIGS. 15A-G and FIGS. 16A-K, respectively). Moreover, the presentinventors were successful in in-planta co-expression of two heterologousgenes (e.g. DsRed and GFP) and specifically in different plantcompartments (e.g. cytosol, chloroplasts or nucleus) using viral vectorsof some embodiments of the invention (see e.g. FIGS. 17A-D and 18A-L).Importantly, the present inventors have generated zinc finger nucleases(ZFNs) which specifically bind and cleave petunia non-coding targetsequences (FIGS. 20-22), petunia phytoene desaturase (PDS) genomicsequences (FIGS. 23 and 28B) or petunia flavanone 3 beta-hydroxylase(FHT) genomic sequences. Accordingly, these chimeric nucleases and viralvectors may serve as powerful tools in the field of agriculturetransgenic technologies.

Thus, according to one aspect of the present invention there is provideda method of generating genotypic variation in a genome of a plant. Themethod comprising introducing into the plant at least one viralexpression vector encoding at least one chimeric nuclease whichcomprises a DNA binding domain, a nuclease and a nuclear localizationsignal, wherein the DNA binding domain mediates specific targeting ofthe nuclease to the genome of the plant, thereby generating genotypicvariation in the genome of the plant.

As used herein the term “plant” refers to whole plants, portions thereof(e.g., leaf, root, fruit, seed) or cells isolated therefrom (homogeneousor heterogeneous populations of cells).

As used herein the phrase “isolated plant cells” refers to plant cellswhich are derived from disintegrated plant cell tissue or plant cellcultures.

As used herein the phrase “plant cell culture” refers to any type ofnative (naturally occurring) plant cells, plant cell lines andgenetically modified plant cells, which are not assembled to form acomplete plant, such that at least one biological structure of a plantis not present. Optionally, the plant cell culture of this aspect of thepresent invention may comprise a particular type of a plant cell or aplurality of different types of plant cells. It should be noted thatoptionally plant cultures featuring a particular type of plant cell maybe originally derived from a plurality of different types of such plantcells.

Any commercially or scientifically valuable plant is envisaged inaccordance with these embodiments of the invention. A suitable plant foruse with the method of the invention can be any monocotyledonous ordicotyledonous plant including, but not limited to, maize, wheat,barely, rye, oat, rice, soybean, peanut, pea, lentil and alfalfa,cotton, rapeseed, canola, pepper, sunflower, potato, tobacco, tomato,lettuce, mums, arabidopsis, broccoli, cabbage, beet, quinoa, spinach,cucumber, squash, watermelon, beans, hibiscus, okra, apple, rose,strawberry, chile, garlic, onions, sorghum, eggplant, eucalyptus, pine,a tree, an ornamental plant, a perennial grass and a forage crop,coniferous plants, moss, algae, as well as other plants listed in WorldWide Web (dot) nationmaster (dot) com/encyclopedia/Plantae.

Accordingly, plant families may comprise Alliaceae, Amaranthaceae,Amaryllidaceae, Apocynaceae, Asteraceae, Boraginaceae, Brassicaceae,Campanulaceae, Caryophyllaceae, Chenopodiaceae, Compositae, Cruciferae,Cucurbitaceae, Euphorbiaceae, Fabaceae, Gramineae, Hyacinthaceae,Labiatae, Leguminosae-Papilionoideae, Liliaceae, Linaceae, Malvaceae,Phytolaccaceae, Poaceae, Pinaceae, Rosaceae, Scrophulariaceae,Solanaceae, Tropaeolaceae, Umbelliferae and Violaceae.

Such plants include, but are not limited to, Allium cepa, Amaranthuscaudatus, Amaranthus retroflexus, Antirrhinum majus, Arabidopsisthaliana, Arachis hypogaea, Artemisia sp., Avena sativa, Bellisperennis, Beta vulgaris, Brassica campestris, Brassica campestris ssp.Napus, Brassica campestris ssp. Pekinensis, Brassica juncea, Calendulaofficinalis, Capsella bursa-pastoris, Capsicum annuum, Catharanthusroseus, Chemanthus cheiri, Chenopodium album, Chenopodium amaranticolor,Chenopodium foetidum, Chenopodium quinoa, Coriandrum sativum, Cucumismelo, Cucumis sativus, Glycine max, Gomphrena globosa, Gossypiumhirsutum cv. Siv'on, Gypsophila elegans, Helianthus annuus, Hyacinthus,Hyoscyamus niger, Lactuca sativa, Lathyrus odoratus, Linumusitatissimum, Lobelia erinus, Lupinus mutabilis, Lycopersiconesculentum, Lycopersicon pimpinellifolium, Melilotus albus, Momordicabalsamina, Myosotis sylvatica, Narcissus pseudonarcissus, Nicandraphysalodes, Nicotiana benthamiana, Nicotiana clevelandii, Nicotianaglutinosa, Nicotiana rustica, Nicotiana sylvestris, Nicotiana tabacum,Nicotiana edwardsonii, Ocimum basilicum, Petunia hybrida, Phaseolusvulgaris, Phytolacca Americana, Pisum sativum, Raphanus sativus, Ricinuscommunis, Rosa sericea, Salvia splendens, Senecio vulgaris, Solanumlycopersicum, Solanum melongena, Solanum nigrum, Solanum tuberosum,Solanum pimpinellifolium, Spinacia oleracea, Stellaria media, SweetWormwood, Trifolium pratense, Trifolium repens, Tropaeolum majus,Tulipa, Vicia faba, Vicia villosa and Viola arvensis. Other plants thatmay be infected include Zea maize, Hordeum vulgare, Triticum aestivum,Oryza sativa and Oryza glaberrima.

According to a specific embodiment of the present invention, the plantcomprises a Petunia hybrida.

According to another specific embodiment of the present invention, theplant comprises a Nicotiana tabacum.

As used herein the phrase “genotypic variation” refers to a process inwhich a nucleotide or a nucleotide sequence (at least 2 nucleotides) isselectively altered or mutated at a predetermined genomic site, alsotermed as mutagenesis. The genomic site may be coding or non-coding(e.g., promoter, terminator, splice site, polyA) genomic site. Thisalteration can be a result of a deletion of nucleic acid(s), arandomized insertion of nucleic acid(s), introduction of a heterologousnucleic acid carrying a desired sequence, or homologous recombinationfollowing formation of a DNA double-stranded break (DSB) in the targetgene. Genotypic variation according to the present teachings may betransient as explained in further detail hereinbelow. Genotypicvariation in accordance with the present teachings is typically effectedby the formation of DSBs, though the present invention also contemplatesvariation of a single strand. Genotypic variation may be associated withphenotypic variation. The sequence specific or site directed nature ofthe present teachings thus may be used to specifically design phenotypicvariation.

As mentioned hereinabove, the method according to this aspect of thepresent invention is effected by introducing into the plant at least oneviral expression vector encoding at least one chimeric nuclease whichcomprises a DNA binding domain, a nuclease and a nuclear localizationsignal.

As used herein the phrase “chimeric nuclease” refers to a syntheticchimeric polypeptide which forms a single open reading frame andmediates DNA cleavage in a sequence specific manner.

As used herein the phrase “DNA binding domain” refers to a native orsynthetic amino acid sequence such as of a protein motif that binds todouble- or single-stranded DNA with affinity to a specific sequence orset thereof (i.e. target site).

In generating chimeric nucleases any DNA binding domain that recognizesthe desired DNA binding sequence with sufficient specificity may beemployed.

Examples of DNA binding domains include, but are not limited to,helix-turn-helix (pfam 01381), leucine zipper (ZIP) domain, winged helix(WH) domain, winged helix turn helix domain (wHTH), helix-loop-helix andzinc finger domain.

Thus, a variety of such DNA binding domains are known in the art. In anexemplary embodiment of the present invention, the DNA binding domain isa zinc finger binding domain (e.g., pfam00096).

The zinc finger domain is 30 amino acids long and consists of arecognition helix and a 2-strand beta-sheet. The domain also containsfour regularly spaced ligands for Zinc (either histidines or cysteines).The Zn ion stabilizes the 3D structure of the domain. Each fingercontains one Zn ion and recognizes a specific triplet of DNA basepairs.

Zinc finger domains can be engineered to bind to a predeterminednucleotide sequence. Each individual zinc finger (e.g. Cys2/His2)contacts primarily three consecutive base pairs of DNA in a modularfashion [Pavletich et al., Science (1991) 252:809-817; Berg et al.,Science (1996) 271:1081-1085]. By manipulating the number of zincfingers and the nature of critical amino acid residues that contact DNAdirectly, DNA binding domains with novel specificities can be evolvedand selected [see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. USA(1992) 89:7345-7349; Rebar et al., Science (1994) 263:671-673; Greismanet al., Science (1997) 275:657-661; Segal et al., Proc. Natl. Acad. Sci.USA (1999) 96:2758-2763]. Hence, a very wide range of DNA sequences canserve as specific recognition targets for zinc finger proteins. Chimericnucleases with several different specificities based on zinc fingerrecognition have been previously disclosed [see for example, Huang etal., J. Protein Chem. (1996) 15:481-489; Kim et al., Biol. Chem. (1998)379:489-495].

Various methods for designing chimeric nucleases with varied DNA bindingdomains are known in the art. In one embodiment the DNA binding domaincomprises at least one, at least two, at least 3, at least 4, at least 5at least 6 zinc finger domains, binding a 3, 6, 9, 12, 15, or 18nucleotide sequence, respectively. It will be appreciated by the skilledartisan that the longer the recognition sequence is, the higher thespecificity that will be obtained.

Specific DNA binding zinc fingers can be selected by using polypeptidedisplay libraries. The target site is used with the polypeptide displaylibrary in an affinity selection step to select variant zinc fingersthat bind to the target site. Typically, constant zinc fingers and zincfingers to be randomized are made from any suitable C2H2 zinc fingersprotein, such as SP-1, SP-1C, TFIIIA, GLI, Tramtrack, YY1, or ZIF268[see, e.g., Jacobs, EMBO J. 11:4507 (1992); Desjarlais & Berg, Proc.Natl. Acad. Sci. U.S.A. 90:2256-2260 (1993)]. The polypeptide displaylibrary encoding variants of a zinc finger protein comprising therandomized zinc finger, one or more variants of which will be selected,and, depending on the selection step, one or two constant zinc fingers,is constructed according to the methods known to those in the art.Optionally, the library contains restriction sites designed for ease ofremoving constant zinc fingers, and for adding in randomized zincfingers. Zinc fingers are randomized, e.g., by using degenerateoligonucleotides, mutagenic cassettes, or error prone PCR. See, forexample, U.S. Pat. Nos. 6,326,166, 6,410,248, and 6479626.

Zinc fingers can also be selected by design. A designed zinc fingerprotein is a protein not occurring in nature whose design/compositionresults principally from rational criteria. Rational criteria for designinclude application of substitution rules and computerized algorithmsfor processing information in a database storing information of existingZFP designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496.

As illustrated in Example 1 hereinbelow, two sets of chimeric nucleaseswere designed according to the present teachings each set capable offorming DSBs in specific target sequences of Petunia DNA. Initially, DNAbinding sequences were identified in Petunia plants which were suitablefor recognition and cleavage by chimeric nucleases. These DNA bindingsequences were non-coding, non-repetitive sequences: P25-TS1 (SEQ ID NO:10), P25-TS2 (SEQ ID NO: 11), P36-TS1 (SEQ ID NO: 12) and P36-TS2 (SEQID NO: 13). Next, chimeric nucleases were designed each comprising 3zinc fingers. As illustrated in FIGS. 21-22, these zinc fingersdesignated P1-25-ZFN1, P1-25-ZFN2, P1-36-ZFN1 and P1-36-ZFN2 (SEQ IDNOs: 35, 36, 37, or 38, respectively) specifically bound and cleaved theabove mentioned Petunia target sites.

Furthermore, as illustrated in Example 1 hereinbelow, according to thepresent teachings, chimeric nucleases were designed capable of formingspecific DSBs in Petunia phytoene desaturase (PDS) genomic sequences orin Petunia flavanone 3 beta-hydroxylase (FHT) genomic sequences.Initially, DNA binding sequences for PDS and FHT were identified inPetunia plants which were suitable for recognition and cleavage by suchchimeric nucleases (see FIGS. 28A and 29, respectively). The DNA bindingsequences for PDS specific zinc fingers were identified: PDS-ZFN1 (SEQID NO: 140) and PDS-ZFN2 (SEQ ID NO: 141) and the chimeric nucleaseswere designed (SEQ ID NOs: 71 and 73, respectively) which specificallybound and cleaved the PDS Petunia target sites. Likewise, the DNAbinding sequences for FHT specific zinc fingers were identified:FHT-ZFN1 (SEQ ID NO: 142) and FHT-ZFN2 (SEQ ID NO: 143) and the chimericnucleases were designed (SEQ ID NOs: 75 and 77, respectively) whichspecifically bound and cleaved the FHT Petunia target sites.

According to an embodiment of the present invention the zinc fingerbinding domain comprises a nucleic acid sequence as set forth in SEQ IDNOs. 17, 18, 19, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

Preferably, the chimeric nucleases of this aspect of the presentinvention comprise separate domains for DNA binding and for DNAcleavage, such that DNA cleavage is sequence specific.

As used herein the phrase “sequence specific” refers to a distinctchromosomal location at which a double stranded break (cleavage) isintroduced. Without being bound by theory, it is believed that theformation of DSB induces a cellular repair mechanism which typicallyleads to highly efficient recombinational events at that locus.

As used herein the term “nuclease” refers to any polypeptide, or complexcomprising a polypeptide, that can generate a strand break in genomicDNA (i.e. comprises DNA cleavage activity). Examples of nucleases whichmay be used in accordance with the present teachings include restrictionenzymes, topoisomerases, recombinases, integrases and DNAses.

It will be appreciated that the nuclease utilized by the presentinvention may comprise any non-specific DNA cleavage domain, forexample, a type II restriction endonuclease such as the cleavage domainof the FokI restriction enzyme (GenBank accession number J04623). FokIrestriction enzymes which generally have separate DNA cleavage and DNAbinding domains are suitable for construction of the chimeric nucleases.Thus, according to an embodiment of this aspect, the chimeric nucleasesare chimeric proteins comprising specific zinc finger binding domainsand the DNA cleavage domain of the FokI restriction enzyme (alsoreferred to herein as the FokI cleavage domain).

In accordance with embodiments of the present invention the chimericnuclease is an isolated polynucleotide comprising a nucleic acidsequence as set forth in SEQ ID NO.31, 32, 33, 34, 70, 72, 74, 76, 84,86 or 88.

In accordance with embodiments of the present invention the chimericnuclease is an isolated polypeptide comprising an amino acid sequence asset forth in SEQ ID NO. 35, 36, 37, 38, 71, 73, 75, 77, 85, 87 or 89.

Since certain nucleases (e.g. FokI) function as dimers, in order tocreate double stranded breaks in the target gene at least two chimericnuclease must be employed. Thus, according to an exemplary embodiment,the chimeric nucleases of the present invention form dimers (e.g., viabinding to both strands of a target sequence). For example, chimericnucleases can form a homodimer between two identical chimeric nucleases(e.g., via binding to two identical DNA binding sequences within atarget sequence). Alternatively, chimeric nucleases can form aheterodimer between two different chimeric nucleases (e.g., via bindingto two different DNA binding sequences within a target sequence, seee.g., FIG. 1). Accordingly, two chimeric nucleases may be employed tocreate a double-stranded break in a target sequence. Consequently, theDNA binding domain of the chimeric nuclease, or two or more conjointlyacting chimeric nucleases may bind a DNA sequence.

Examples of nucleases which can be used according to the presentteachings include, but are not limited to, restriction enzymes includingFokl, Scel, I-CeuI, artificial meganucleases, modified meganucleases,homing nucleases; topoisomerases including DNA gyrase, eukaryotictopoisomerase II, bacterial topoisomerase IV and topoisomerase VI;recombinases including Cre recombinase, Hin recombinase, Rad51/RecA;DNAses including deoxyribonuclease I, deoxyribonuclease II andmicrococcal nuclease; and integrases.

The phrase “DNA-containing organelle” refers to a subcellular,membrane-encapsulated structure, present in all plant cells.

DNA containing organelles include, the mitochondrion, the nucleus, thechloroplast, the proplastid, the etioplast, the chromoplast and theleukoplast, and any subcellular structure which includes DNA molecules.Typically, the DNA is endogenous but in some cases may refer toexogenous DNA such as of a plant pathogen such as a virus. In the lattercase, for example, the DNA-containing organelle is the cytoplasm, inwhich case the chimeric nuclease may not comprise any localizationsignal.

It will be appreciated that generating genotypic variation in plantorganelles other than the nucleus is of particular interest according tosome embodiments of the present invention, as will be detailed infra.Plant organelles (e.g. chloroplast and mitochondria) contain DNA whichis a vital participant in plant biochemical pathways. These organelleshave a wide structural and functional diversity. As such, they are ableto transcribe and translate the information present in their own genomebut are strongly dependent on imported proteins that are encoded in thenuclear genome and translated in the cytoplasm.

For example, the chloroplast performs essential metabolic andbiosynthetic functions of global significance, including photosynthesis,carotenoids and amino acid biosynthesis. Carotenoids are integralconstituents of plants, they are isoprenoids pigments which are involvedin a variety of processes including protection against photooxidativestress (through energy-dissipation of excess light absorbed by theantenna pigments); coloring agents in flowers and fruits to attractpollinators, and precursors for the plant growth hormone abscisic acidand vitamin A [Cunningham and Gantt (1998) Annu Rev Plant Physiol PlantMol Biol 49:557-583]. The carotenoid pigments are synthesized in theplastids of plants where it is derived from the pathways of isoprenoidbiosynthesis (Cunningham and Gantt, supra). Two biosynthetic pathwaysfor isoprenoid biosynthesis are present in plants, the mevalonatepathway found in the cytoplasm and the methylerythritol 4-phosphate(MEP) pathway found only in the plastids. The latter biosynthetic routebeing strongly linked to photosynthesis [Seemann et al. (2006) FEBSLett. 580: 1547-1552].

Moreover, the aromatic amino acid phenylalanine may be synthesized inchloroplasts from the intermediate prephenate: via arogenate by theactivity of prephenate aminotransferase or via phenylpyruvate by theactivity of prephenate dehydratase [Jung et al. (1986). Proc. Natl.Acad. Sci. 83: 7231-7235; Rippert et al. (2009) Plant Physiol. 149(3):1251-1260].

Furthermore, the nitrite reductase and acetolactate synthetase activityof the cell is also located in the plastids. The plastids were found tocontain only part of the total glutamine synthetase, aspartateaminotransferase, and triosephosphate dehydrogenase activity in the cell[Miflin B (1974) Plant Physiol. 54(4): 550-555]. The chloroplast is alsoinvolved in methionine metabolism in plants, chloroplasts are autonomousfor de novo methionine synthesis and can import S-adenosylmethioninefrom the cytosol [Ravanel et al. (2004). J. Biol. Chem. 279 (21):22548-22557].

Similarly, the mitochondria comprise key roles in cellular metabolicpathways, catalyzing one or several steps in these pathways (e.g. thesynthesis of the vitamins folate and biotin, of the non-vitamin coenzymelipoate, of the cardiolipin diphosphatidylglycerol. Although themitochondria lack acetyl-CoA carboxylase, it contains the enzymaticequipment necessary to transform malonate into the two main buildingunits for fatty acid synthesis: malonyl- and acetyl-acyl carrier protein(ACP).

Cytoplasmic male sterility (CMS) in plants, characterized by thesuppression of the production of viable pollen and by the non-Mendelianinheritance of this trait, is associated with mitochondrial dysfunction.The genetic determinants for cytoplasmic male sterility reside in themitochondrial genome. CMS phenotype essentially affects the pollenproducing organs due to the high requirement of energy by this tissue.Thus, a mitochondrial dysfunction will dramatically affect pollenproduction while other plant organs may overcome the consequences ofmitochondrial dysfunction.

As used herein the phrase “localization domain” refers to a localizationsignal which facilitates the transport of the chimeric nucleases to theDNA-containing organelle.

The localization signal can be for example, a nuclear localizationsignal (NLS), such as a short predominantly basic amino acid sequence,which is recognized by specific receptors at the nuclear pores. In otherexemplary embodiments, the localization signal for a DNA containingorganelle can be a mitochondrial localization signal (MLS) or achloroplast localization signal (CLS).

Essentially any NLS may be employed, whether synthetic or a naturallyoccurring NLS, as long as the NLS is one that is compatible with thetarget cell (i.e. plant cell).

Although nuclear localization signals are discussed herewith, thepresent teachings are not meant to be restricted to these localizationsignals, as any signal directed to a DNA-containing organelle isenvisaged by the present teachings. Such signals are well known in theart and can be easily retrieved by the skilled artisan.

Nuclear localization signals which may be used according to the presentteachings include, but are not limited to, SV40 large T antigen NLS,acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcriptionrepressor Matα2 and the complex signals of U snRNPs, tobacco NLS andrice NLS.

Mitochondrion localization signals which may be used according to thepresent teachings include, but are not limited to the transition signalsof, Beta ATPase subunit [cDNAs encoding the mitochondrial pre-sequencesfrom Nicotiana plumbaginifolia β-ATPase (nucleotides 387-666)],Mitochondrial chaperonin CPN-60 [cDNAs encoding the mitochondrialpre-sequences from Arabidopsis thaliana CPN-60 (nucleotides 74-186] andCOX4 [the first 25 codons of Saccharomyces cerevisiae COX4 which encodesthe mitochondrial targeting sequence].

According to a specific embodiment of the present invention, thelocalization signal may comprise a mitochondria localization signal,such as the signal peptide of the ATPase beta subunit (ATP-β) (SEQ IDNO: 139).

Chloroplast localization signals which may be used according to thepresent teachings include, but are not limited to the transition signalsof the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit(ats1A) associated transit peptide, the transition signal of LHC II, aswell as the N-terminal regions of A. thaliana SIG2 and SIG3 ORFs. Seealso http://wwwdotspringerlinkdotcom/content/p65013h263617795/.

Alternatively, the chloroplast localization sequence (CLS) may bederived from a viroid [Evans and Pradhan (2004) US 2004/0142476 A1]. Theviroid may be an Avsunviroiae viroid, for example, an Avocado SunblotchViroid (ASBVd), a Peach Latent Mosaic Virus (PLMVd), a ChrysanthemumChlorotic Mottle Viroid (CChMVd) or an Eggplant Latent Viroid (ELVd).

According to a specific embodiment of the present invention, thelocalization signal may comprise a chloroplast localization signal, suchas the transit peptide ribulose-1,5-bisphospate carboxylase smallsubunit (Rssu) (SEQ ID NO: 138).

For efficient gene targeting, the DNA binding domain of the presentinvention needs to be coupled to the nuclease as to permit DNA cleavagewithin a workable proximity of the target sequence. A workable proximityis any distance that still facilitates the sequence targeting.Optionally, the DNA binding domain overlaps the target sequence or maybind within the target sequence.

Recombinant DNA technology is typically used to generate the chimericnucleases of the present invention [see Example 1 of the Examplessection which follows and Sambrook et al., Eds., Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor University Press, NewYork (1989); Ausubel et al., Eds., Current Protocols in MolecularBiology, John Wiley & Sons, New York (1998); and Maeder, et al. (2008)Mol Cell 31:294-301, as well as other references which are providedhereinbelow].

Qualifying chimeric nucleases thus generated for specific targetrecognition can be effected using methods which are well known in theart.

A method for designing a chimeric nuclease for use in gene targeting mayinclude a process for testing the toxicity of the chimeric nuclease on acell. Such a process may comprise expressing in the cell, or otherwiseintroducing into a cell, the chimeric nuclease and assessing cell growthor death rates by comparison against a control. The tendency of achimeric nuclease to cleave at more than one position in the genome maybe evaluated by in vitro cleavage assays, followed by electrophoresis(e.g. pulsed field electrophoresis may be used to resolve very largefragments) and, optionally, probing or Southern blotting (see Example 5in the Examples section which follows). In view of the presentdisclosure, one of ordinary skill in the art may devise other tests forcleavage specificity.

In one specific embodiment, the present invention provides two sets ofchimeric nucleases: P1-25-ZFN1 and P1-25-ZFN2 (shown in SEQ ID NO: 35and 36, respectively) for gene targeting at the P1-25 site 1 (SEQ ID NO:10) and P1-25 site 2 (SEQ ID NO: 11) of Petunia, respectively; andP1-36-ZFN1 and P1-36-ZFN2 (shown in SEQ ID NO: 37 and 38, respectively)for gene targeting at the P1-36 site 1 (SEQ ID NO: 12) and P1-36 site 2(SEQ ID NO: 13) of Petunia. In particular, P1-25-ZFN1 and P1-25-ZFN2 canform a dimer and P1-36-ZFN1 and P1-36-ZFN2 can form a dimer forgenerating specific double stranded breaks in Petunia target genes.

In another embodiment of the present invention there is provided a setof PDS chimeric nucleases: PDS-ZFN1 and PDS-ZFN2 (shown in SEQ ID NO: 71and 73, respectively) for gene targeting at the PDS site 1 (SEQ ID NO:140) and PDS site 2 (SEQ ID NO: 141) of Petunia, respectively. Thesechimeric nucleases can form a dimer for generating specific doublestranded breaks in Petunia PDS gene.

In another embodiment of the present invention there is provided a setof FHT chimeric nucleases: FHT-ZFN1 and FHT-ZFN2 (shown in SEQ ID NO: 75and 77, respectively) for gene targeting at the FHT site 1 (SEQ ID NO:142) and FHT site 2 (SEQ ID NO: 143) of Petunia, respectively. Thesechimeric nucleases can form a dimer for generating specific doublestranded breaks in Petunia FHT gene.

As mentioned hereinabove, the chimeric nuclease is introduced into theplant target using a viral expression vector, which is typically usedfor mediating transient transformation, systemically spreading withinthe plant such as through the meristem infection.

Thus, according to another aspect of the present invention there isprovided a plant viral expression vector comprising a nucleic acidsequence encoding at least one chimeric nuclease which comprises a DNAbinding domain, a nuclease and optionally a localization signal.

As used herein a plant viral expression vector refers to a nucleic acidvector including a DNA vector (e.g., a plasmid), a RNA vector, virus orother suitable replicon (e.g., viral vector) encoding for viral genes orparts of viral genes.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO 87/06261

Other viruses which may be useful in transformation of plant hostsinclude tobacco rattle virus (TRV) and its related viruses. TRV is knownfor its ability to infect meristematic tissues, it comprises a broadhost range and different strain isolates. For example strain N5,obtained from narcissus, causes severe necrosis in Nicotiana clevelandii[Harrison et al. (1983) Ann. appl. Biol., 102:331-338]. The hypochoerismosaic virus (HMV), which is serologically related to TRV [Uhde et al.(1998) Archives of Virology 143:1041-1053], infects the Asteraceaefamily of plants [Brunt and Stace-Smith (1978) Ann. appl. Biol.90:205-214]. The tobacco rattle virus strain TCM, originally obtainedfrom tulip, is serologically closely related to the Dutch serotype ofPea early-browning virus [Robinson et al., J. Gen. Virol. (1987)68:2551-2561). Furthermore, there are also monocotyledons speciessusceptible to TRV, as for example Avena sativa (family Poaceae) [Cadmanand Harrison, Ann. appl. Biol. (1959) 47:542-556].

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by replicating the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous nucleic acid sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931, Dawson, W.O. et al. (1989). A tobacco mosaic virus-hybrid expresses and loses anadded gene. Virology 172, 285-292; French, R. et al. (1986) Science 231,1294-1297; and Takamatsu, N. et al. (1990). Production of enkephalin intobacco protoplasts using tobacco mosaic virus RNA vector. FEBS Lett269, 73-76.

In one embodiment, a plant viral nucleic acid is provided in which thenative coat protein coding sequence has been deleted from a viralnucleic acid, a non-native plant viral coat protein coding sequence anda non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral nucleic acid, andensuring a systemic infection of the host by the recombinant plant viralnucleic acid, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native nucleic acid sequencewithin it, such that a protein is produced. The recombinant plant viralnucleic acid may contain one or more additional non-native subgenomicpromoters. Each non-native subgenomic promoter is capable of replicatingor expressing adjacent genes or nucleic acid sequences in the plant hostand incapable of recombination with each other and with nativesubgenomic promoters. Non-native (foreign, heterologous) nucleic acidsequences may be inserted adjacent the native plant viral subgenomicpromoter or the native and a non-native plant viral subgenomic promotersif more than one nucleic acid sequence is included. The non-nativenucleic acid sequences are replicated or expressed in the host plantunder control of the subgenomic promoter to produce the desiredproducts.

In a second embodiment, a recombinant plant viral nucleic acid isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral nucleic acid isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of replicating or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that said sequences are replicated or expressed in the host plantunder control of the subgenomic promoters to produce the desiredproduct.

In a fourth embodiment, a recombinant plant viral nucleic acid isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral nucleic acid to produce a recombinant plantvirus. The recombinant plant viral nucleic acid or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral nucleic acid is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(isolated nucleic acid) in the host to produce the desired protein i.e.,the chimeric nuclease and optionally other heterologous coding ornon-coding nucleic acid sequences.

A viral expression vector comprising a nucleic acid encoding a chimericnuclease is operably linked to one or more transcriptional regulatorysequences whereby the coding sequence is under the control oftranscription signals to permit production or synthesis of the chimericnuclease. Such transcriptional regulatory sequences include promotersequences, enhancers, and transcription binding sites.

Promoters which are known or found to cause transcription of a foreigngene in plant cells can be used in the present invention. Such promotersmay be obtained from plants or viruses and include, but are not limitedto, the 35S promoter of cauliflower mosaic virus (CaMV) (includesvariations of CaMV 35S promoter, e.g. promoters derived by means ofligations with operator regions, random or controlled mutagenesis,etc.), promoters of seed storage protein genes such as Zma10 Kz orZmag12 (maize zein and glutelin genes, respectively), light-induciblegenes such as ribulose bisphosphate carboxylase small subunit (rbcS),stress induced genes such as alcohol dehydrogenase (Adhl), or“housekeeping genes” that express in all cells (such as Zmaact, a maizeactin gene). For added control, the chimeric nuclease may be under thecontrol of an inducible promoter.

In one embodiment the plant viral expression vector is a tobacco rattlevirus (TRV) expression vector.

TRV-based expression vectors have been described in for example U.S.Pat. No. 7,229,829.

TRV is a positive strand RNA virus with a bipartite genome, hence thegenome is divided into two positive-sense, single-stranded RNAs, thatmay be separately encapsidated into viral particles. The two TRV genomicRNA vectors used by the present invention are referred to herein aspTRV1 (GeneBank Accession No: AF406990) and pTRV2 (GeneBank AccessionNo: AF406991), wherein pTRV1 encodes polypeptides that mediatereplication and movement in the host plant while pTRV2 encodes coatproteins.

In certain embodiments, the nucleic acid sequence of pTRV2 is devoid of2b sequence (SEQ ID NO: 43). Generating a pTRV2 vector devoid of the 300by of the RNA2 2b gene was carried out by removal from the originalvector by digestion with PvuII and EcoRI (see FIGS. 6A-B). The resultantplasmid (pTRV2Δ2b) was identical to the original pTRV2 but lacking the2b sequence (see Example 1, hereinbelow). According to the presentteachings, pTRV2 vectors without the 2b region are much more efficientin gene expression in meristematic tissues (see Example 2, hereinbelow).

In certain embodiments, modification to pTRV2 vector comprises additionof an enhancer. Any enhancer can be inserted into the viral expressionvector to enhance transcription levels of genes. For example, a Ωenhancer (SEQ ID NOs: 44 or 47) can be cloned into the pTRV2 vectors ofthe present invention.

Alternatively, the viral vector of the present invention may be based onTRV related viruses (e.g. tobacco rattle virus strain N5, HMV, ortobacco rattle virus strain TCM).

The selection of the vector may be dependent on the target plant such asmonocots. The modified wheat streak mosaic virus (WSMV) has beenpreviously shown to express NPT II and β-glucuronidase (GUS) in monocots(e.g. wheat, barley, oat and maize) [Choi et al., Plant J. (2000)23:547-555; Choi et al., J Gen Virol (2002) 83:443-450; Choi et al., J.Gen. Virol. (2005) 86:2605-2614]. The work of Choi et al. demonstratedthe placement of the foreign genes between the nuclear inclusion b (NIb)and coat protein (CP). For better expression and activity in infectedwheat, GUS was inserted immediately downstream of the P1 cleavage siteand up stream to HC-Pro of the wheat streak mosaic virus (WSMV)polyprotein ORF. Systemic infection and GUS expression was demonstratedupon inoculation of plants with WSMV in vitro.

The present invention contemplates a viral expression vector comprisingat least two heterologous polypeptide sequences.

As used herein the term “heterologous sequence” refers to a sequencethat is not normally part of an RNA2 of a naturally occurring TRV. Incertain embodiments, a heterologous sequence is a chimeric nuclease asdescribed in detail hereinabove. In certain embodiments, a heterologoussequence is a sequence of interest, such as a plant gene for expressionin a plant cell of a heterologous polypeptide. Such plant genes mayinclude, but are not limited to, genes encoding a reporter polypeptide,an antiviral polypeptide, a viral moiety, an antifungal polypeptide, anantibacterial polypeptide, an insect resistance polypeptide, a herbicideresistance polypeptide, a biotic or abiotic stress tolerancepolypeptide, a pharmaceutical polypeptide, a growth inducingpolypeptide, and a growth inhibiting polypeptide. In certainembodiments, the viral vector comprises both chimeric nucleases and asequence of interest.

As part of the pTRV vector, the heterologous sequences may compriseseparate sub genomic promoters (sgPs), thus may comprise two separatesgPs (e.g. SEQ ID NO: 45 and SEQ ID NO: 48) for replication of theheterologous sequences.

In certain embodiments, the at least two heterologous polypeptidesequences within the viral vector are separated by nucleic acid sequenceencoding a cleavage domain. Such a cleavage domain may comprise anycleavage domain known in the art, as for example a T2A-like proteinsequence (SEQ ID NOs: 40 and 52).

It will be appreciated that the nucleic acid sequence of the twoheterologous polypeptide sequences separated by a cleavage domain may beas set forth in SEQ ID NOs: 84, 86 or 88.

It will be appreciated that the amino acid sequence of the twoheterologous polypeptide sequences separated by a cleavage domain may beas set forth in SEQ ID NOs: 85, 87 or 89.

Generally, when introduced into a host plant cell, a pTRV vectorprovides expression of the heterologous sequence(s) and may also provideexpression of other TRV sequences, such as a viral coat protein.

pTRV vectors of the present invention may express a reporter gene sothat transformed cells can be identified. Exemplary reporter genes thatmay be expressed include, but are not limited to, GUS and GFP.

It will be appreciated that two viral expression vectors may beintroduced into the same plant cell. These viral vectors may beintroduced in the plant cell concomitantly or at separate times. Suchviral expression vectors may comprise the same type of vector encodingdifferent heterologous sequences, or alternatively may comprise twodifferent types of vectors (e.g. BV vector and TRV vector, mitovirusvector and TRV vector, TRV1 and TRV2 vectors). For example, pTRV1 andpTRV2 vectors can be introduced concomitantly, as for example at a 1:1ratio, to enable expression of viral genes in plant cells. Likewise, onevector may comprise the chimeric nuclease/s and another vector maycomprise a heterologous gene of interest (as described in detailhereinabove).

It will be appreciated that in order to introduce the heterologous geneof interest (i.e. foreign DNA) into different DNA containing organelles(e.g. nucleus, chloroplast and mitochondria), different types of vectorsmay be implemented.

Thus, vectors for delivery of foreign DNA may be based on theGeminivirus Abutilon mosaic virus (AbMV), a member of the Begomovirusgenus. The AbMV viral DNA has been detected in plastids [Groning et al.,(1987) PNAS USA 84: 8996; Groning et al. (1990) Mol. Gen. Gene. 220:485; Horns & Jeske, (1991) Virol. 181: 580].

The viral vector of the present invention may also be based on the genusMitovirus, family Narnaviridae such as H. mompa mitovirus 1-18(HmMV1-18) or O. novo-ulmi mitovirus 6 (OnuMV6). The HmMV1-18 viraldsRNA has been detected in mitochondria [Osaki et al (2005) Virus res.107, 39-46; Cole et al (2000) Virol. 268, 239-243].

Other DNA virus based vectors that are envisioned by the presentinvention include, for example, Geminiviridae, Caulimoviridae andBadnaviridae.

For example, Geminiviridae contain circular covalently closedsingle-stranded (ss) DNA (−2.8 Kbp) genomes, packaged within twinned(so-called geminate) particles.

The sequences regulating DNA replication and transcriptional activityare located in the intergenic regions (IR). The invariant TAATATT_ACsequence is located in the LIR (in mastreviruses), IR (in curtoviruses)and CR (in begomoviruses) and contains the initiation site ofrolling-circle DNA replication. The geminivirus replication cycle can besubdivided in several functionally distinct stages. Early during theinfection process, viral particles are injected by the insect vector,presumably uncoated, and the viral genome is transported into the hostcell nucleus where all later stages occur: conversion of circular ssDNAinto covalently closed circular dsDNA intermediates, rolling-circlereplication (RCR), production of circular ssDNA genomes forencapsidation [Gutierrez (1999) Cell. Mol. Life Sci. 56 313-329].

Geminiviruses are divided into four genera on the basis of their genomeorganizations and biological properties [Fauquet et al (2003) Arch Virol148: 405-421]. Mastreviruses (e.g. Maize streak virus, Panicum streakvirus, Sugarcane streak virus, Sugarcane streak Egypt virus, Sugarcanestreak Reunion virus, Digitaria streak virus, etc) have monopartitegenomes and are transmitted by leafhopper to monocotyledonous plants.Curtoviruses (e.g. Beet curly top virus) have monopartite genomesdistinct from those of the mastreviruses and are transmitted byleafhopper vectors to dicotyledonous plants. Topocuviruses (e.g. Tomatopseudo-curly top virus) have monopartite genomes which are transmittedby a treehopper vector to dicotyledonous plants. Begomoviruses (e.g.Bean golden yellow mosaic virus, Tomato yellow leaf curl virus, Abutilonmosaic virus, Tobacco leaf curl virus, African cassava mosaic virus,Mung bean yellow mosaic virus) have bipartite genomes (although numerousbegomoviruses with a monopartite genome also occur) and are transmittedby the whitefly Bemisia tabaci to dicotyledonous plants.

Caulimovirus particles contain a single molecule of dsDNA (˜8 kbp).Caulimoviruses usually infect hosts systemically; they are found in mostmesophyll, parenchyma and epidermal cells and sometimes in phloem sievetubes and tracheids. Members of the genus include e.g. Cauliflowermosaic virus (CaMV), Soybean chlorotic mottle (SoyCMV), Cassava veinmosaic (CVMV), Petunia vein clearing (PVCV), Rice tungro bacilliformvirus (RTBV).

It will be appreciated that the universal vector IL-60 and auxiliaryconstructs, which has been recently described [WO 2007/141790] may alsobe used by the present invention. This vector which is, in fact, adisarmed form of Tomato yellow leaf curl virus (begomovirus), is appliedas a double-stranded DNA [Peretz et al (2007) Plant Physiology145:1251-1263]. With IL-60 as the disarmed helper “virus”,transactivation occurs, resulting in an inducible expression/silencingsystem.

In order to direct the vectors containing the foreign DNA into specificDNA containing organelles, a nuclear localization signal (NLS),chloroplast localization signal (CLS) or mitochondria localizationsignal (MLS) may be introduced inframe to the heterologous sequence (asis described in further detail hereinabove).

To achieve transformation of plant cells or the whole plant, the viralexpression vectors of the present invention can be introduced into thehost cell by any method known in the art. For example, transienttransformation can be achieved by Agrobacterium-mediated gene transfer,by direct DNA transfer methods, by viral infection (i.e. using themodified plant viruses) or by nematodes, by infiltration, by vacuum, byelectroporation or by bombardment.

Agrobacterium-mediated gene transfer as disclosed herein (see forexample, Example 1 hereinbelow) includes the use of plasmid vectors thatcontain defined DNA segments. For example, the present invention teachesthe use of Agrobacterium tumefaciens (strain AGLO and EHA-105)transformed with pTRV1, pTRV2 and pTRV2 derivatives containing plasmidsas was previously described [see e.g. Liu et al., Plant J (2002) 30:415-429]. Methods of inoculation of the plant tissue vary depending uponthe plant species and the Agrobacterium delivery system. A widely usedapproach is the leaf-disc procedure, which can be performed with anytissue explant that provides a good source for initiation of whole-plantdifferentiation [Horsch, R. B. et al. (1988). “Leaf disctransformation.” Plant Molecular Biology Manual AS, 1-9, Kluwer AcademicPublishers, Dordrecht]. A supplementary approach employs theAgrobacterium delivery system in combination with vacuum infiltration.The Agrobacterium system is especially useful for in the creation oftransgenic dicotyledenous plants. See: Klee, H. J. et al. (1987). AnnuRev Plant Physiol 38, 467-486; Klee, H. J. and Rogers, S. G. (1989).Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, MolecularBiology of Plant Nuclear Genes, pp. 2-25, J. Schell and L. K. Vasil,eds., Academic Publishers, San Diego, Cal.; and Gatenby, A. A. (1989).Regulation and Expression of Plant Genes in Microorganisms, pp. 93-112,Plant Biotechnology, S. Kung and C. J. Arntzen, eds., ButterworthPublishers, Boston, Mass. The present teachings also discloseAgrobacterium-mediated gene transfer by injection of Agrobacteria intothe plant (e.g. into the exposed shoot surface following removal of theapical meristems) and by leaf infiltration as for example using asyringe without a needle (e.g. Agrobacteria content of the syringe isdischarged into the scratched surface of the leaf, see Example 1 of theexample section which follows).

Direct DNA transfer methods include for example electroporation,microinjection and microparticle bombardment. See, e.g.: Paszkowski, J.et al. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6,Molecular Biology of Plant Nuclear Genes, pp. 52-68, J. Schell and L. K.Vasil, eds., Academic Publishers, San Diego, Cal.; and Toriyama, K. etal. (1988). Bio/Technol 6, 1072-1074 (methods for direct uptake of DNAinto protoplasts). These methods may further be used to direct theforeign DNA containing vectors (as depicted in detail hereinabove) intospecific DNA containing organelles. For example, tobacco protoplastswere electroporated co-transformed with both DNA encoding the nucleaseand donor DNA [Wright et al. (2005) Plant J 44:693-705].

Infection of viral vectors (e.g. pTRV) into plants can also be carriedout by the use of nematodes, including without limitation, N.benthamiana or N. clevelandii (the natural host for TRV). Accordingly,N. benthamiana or N. clevelandii are inoculated with pTRV1, pTRV2 ortheir derivatives prior to subjection to the plants.

Infection of viral vectors into plants may also be effected by virioninfection (as depicted in detail in Example 1, hereinbelow). Virioninfection may be carried out, for example, by first inoculating theusual hosts of the virus (e.g. TRV infection of Petunia) with the viralvector (pTRV1, pTRV2, or its derivatives). About 5 to 21 days postinfection (dpi) plant leaves are collected and the sap is extracted in20 mM phosphate buffer pH=6.8 and a surfactant (e.g. 0-0.03% SilwetL-77) by mortar and pestle. The TRV containing sap is then dripped ontocheesecloth or centrifuged to remove cells debris and following additionof carborundum fine powder (to improve infection) stems and leaves ofyoung (approximately 1 month old) plants are gently scratched. Sapinfection of in-vitro grown plants may also be carried by first passingthe sap through 0.22 μm filter and then stems of tissue culturepropagated plants are injured and infected using syringe and needle orby vacuum. For seeds infection (e.g. monocotyledon), seeds may beincubated with the sap during swelling and sprouting (for approximately1 to 2 weeks).

A transgenic whole plant, callus, tissue or plant cell may be identifiedand isolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the viral expressionvectors. For instance, selection may be performed by growing theengineered plant material on media containing an inhibitory amount ofthe antibiotic or herbicide to which the transforming gene constructconfers resistance. Further, transgenic plants and plant cells may alsobe identified by screening for the activities of any visible markergenes (e.g., GFP or GUS) that may be present on the viral expressionvectors. Such selection and screening methodologies are well known tothose skilled in the art.

Physical and biochemical methods may also be employed to identifytransgenic plants or plant cells containing inserted gene constructs.These methods include, but are not limited to, Southern analysis or PCRamplification, Northern blot, enzymatic assays, protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays. Additional techniques, such as in situhybridization, enzyme staining, and immunostaining, also may be used todetect the presence or expression of the heterologous genes in specificplant organs and tissues. The methods for doing all these assays arewell known to those skilled in the art.

Other references which may be used to implement the teachings of thepresent invention are provided infra: Agrobacterium delivery of a Tiplasmid harboring both the ZFNs and a donor DNA construct [Cai et al.(2009) Plant Mol Biol. Accepted: 14 Dec. 2008].

The above mentioned methods, chimeric nucleases and vectors may be usedfor generating genotypic variation in plants.

The following section provides non-limiting applications for generatingsuch a variation.

Thus, chimeric nucleases of the present invention may be used togenerate a signature of randomly inserted nucleic acids in asequence-specific manner, also referred to herein as tagging. Thissignature may be used as a “genetic mark”. This term is used hereindistinctively from the common term “genetic marker”. While the latterterm refers to naturally occurring genetic variations among individualsin a population, the term genetic mark as used herein specificallyrefers to artificial (man generated), detectable genetic variability,which may be inherited.

The DSB is typically directed into non-coding regions (non open readingframe sequence) so as not to affect the plant's phenotype (e.g. fortagging). However, tagging can also be directed to a coding region. Ahigh quality genetic mark is selected unique to the genome of the plantand endures sequence variation which may be introduced along thegenerations.

For some, e.g., regulatory, purposes it may be desired to markcommercially distributed plants with publicly known marks, so as toenable regulatory authorities to readily identify the mark, so as toidentify the manufacturer, distributor, owner or user of the markedorganism. For other purposes secrecy may be advantageous. The latter istrue, for example, for preventing an attempt to genetically modify thegenetic mark of a supreme event protected by intellectual property laws.

An intellectual property protected organism which is also subject toregulation will therefore be, according to a useful embodiment of thepresent invention, genetically marked by (a) at least one unique DNAsequence which is known in public; and (b) at least one unique DNAsequence that is unknown, at least not as a genetic mark, in public.

To introduce a heterologous sequence (e.g., coding or non-coding), DSBswill first be generated in plant DNA as described herein. It is wellknown those of skill in the art that integration of foreign DNA occurswith high frequency in these DNA brake sites [Salomon et al., EMBO J(1998) 17: 6086-6095; Tzfira et al., Plant Physiol (2003) 133:1011-1023; Tzfira et al., Trends Genet (2004) 20: 375-383, Cai et al.(2009) Plant Mol Biol. Accepted: 14 Dec. 2008]. Once present in thetarget cell, for example on episomal plasmids, foreign DNA may be cutout from the plasmid using the same ZFN used to generate DSBs in theplant DNA. The foreign DNA released from the episomal plasmid will thenbe incorporated into the cell DNA by plant non-homologous end joining(NHEJ) proteins. The DSBs may also lead to enhanced homologousrecombination (HR)-based gene targeting in plant cells (Puchta et al.Proc Natl Acad Sci USA (1996) 93: 5055-5060).

As mentioned, the present teachings can be used to generate genotypicvariation. Thus, the chimeric nucleases of the present teachings can bedesigned to generate DSBs in coding or non-coding regions of a locus ofinterest so as to introduce the heterologous gene of interest. Suchalterations in the plant genome may consequently lead to additions oralterations in plant gene expression (described in detail hereinabove)and in plant phenotypic characteristics (e.g. color, scent etc.).

Additionally chimeric nucleases can be used to generate genotypicvariation by knocking out gene expression. Thus chimeric nucleases canbe designed to generate DSBs in coding or non-coding regions of a locusof interest so as to generate a non-sense or mis-sense mutation.Alternatively, two pairs of chimeric nucleases can be used to cleave outan entire sequence of the genome, thereby knocking out gene expression.

Chimeric nucleases of the present invention may also be used to generatevariability by introducing non-specific mutations into the plant'sgenome. This may be achieved by the use of non-specific DNA restrictasesor Non-stringent Fok1.

As an alternative, the chimeric nucleases of the present invention maybe used to combat infections by plant pathogens.

Thus the present invention envisages a method of treating a plantinfection by a pathogen. The method comprising introducing into theplant at least one expression vector encoding at least one chimericnuclease which comprises a DNA binding domain and a nuclease, whereinthe DNA binding domain mediates targeting of the nuclease to the genomeof the pathogen, thereby of preventing or treating a plant infection bya pathogen.

As used herein a “plant pathogen” refers to an organism, which causes adisease in the infected plant. Organisms that cause infectious diseaseinclude fungi, oomycetes, bacteria, viruses, viroids, virus-likeorganisms, phytoplasmas, protozoa, nematodes and parasitic plants.

Since complete destruction of the DNA of the pathogen is desired, thechimeric nuclease is designed so as to cleave as much sequence sites onthe pathogen's DNA as possible. Thus, repeating sequences may betargeted. Additionally or alternatively a number of distinct sequencesare targeted sufficient to induce degradation of the pathogen's DNA.

According to some embodiments of this aspect of the present invention,the chimeric nuclease is designed to cleave the DNA of the pathogen butnot that of the plant. To this end, the chimeric nuclease is designeddevoid of a localization signal, such that the chimeric nuclease isactive in the cytoplasm which comprises the pathogen's (e.g., virus) DNAbut not that of the plant.

Alternatively, the nuclease may be designed so as to cleave sequenceswhich are specific for the pathogen but are absent from the plant'sgenome. This may be achieved using routine bioinformatics analysis suchas by the use of alignment software e.g., Blast(http://wwwdotncbidotnlmdotnihdotgov/blast/Blastdotcgi).

A non-limiting list of plant viral pathogens which may be targeted usingthe teachings of the present invention include, but are not limited toSpecies: Pea early-browning virus (PEBV), Genus: Tobravirus. Species:Pepper ringspot virus (PepRSV), Genus: Tobravirus. Species: Watermelonmosaic virus (WMV), Genus: Potyvirus and other viruses from thePotyvirus Genus. Species: Tobacco mosaic virus Genus (TMV), Tobamovirusand other viruses from the Tobamovirus Genus. Species: Potato virus XGenus (PVX), Potexvirus and other viruses from the Potexvirus Genus.Thus the present teachings envisage targeting of RNA as well as DNAviruses (e.g. Gemini virus or Bigeminivirus). Geminiviridae viruseswhich may be targeted include, but are not limited to, Abutilon mosaicbigeminivirus, Ageratum yellow vein bigeminivirus, Bean calico mosaicbigeminivirus, Bean golden mosaic bigeminivirus, Bhendi yellow veinmosaic bigeminivirus, Cassava African mosaic bigeminivirus, CassavaIndian mosaic bigeminivirus, Chino del tomaté bigeminivirus, Cotton leafcrumple bigeminivirus, Cotton leaf curl bigeminivirus, Croton yellowvein mosaic bigeminivirus, Dolichos yellow mosaic bigeminivirus,Euphorbia mosaic bigeminivirus, Horsegram yellow mosaic bigeminivirus,Jatropha mosaic bigeminivirus, Lima bean golden mosaic bigeminivirus,Melon leaf curl bigeminivirus, Mung bean yellow mosaic bigeminivirus,Okra leaf-curl bigeminivirus, Pepper hausteco bigeminivirus, PepperTexas bigeminivirus, Potato yellow mosaic bigeminivirus, Rhynchosiamosaic bigeminivirus, Serrano golden mosaic bigeminivirus, Squash leafcurl bigeminivirus, Tobacco leaf curl bigeminivirus, Tomato Australianleafcurl bigeminivirus, Tomato golden mosaic bigeminivirus, TomatoIndian leafcurl bigeminivirus, Tomato leaf crumple bigeminivirus, Tomatomottle bigeminivirus, Tomato yellow leaf curl bigeminivirus, Tomatoyellow mosaic bigeminivirus, Watermelon chlorotic stunt bigeminivirusand Watermelon curly mottle bigeminivirus.

The present invention also envisages a method of generate male sterilityin a plant. The method comprising upregulating in the plant a structuralor functional gene of a mitochondria or chloroplast associated with malesterility by introducing into the plant at least one viral expressionvector encoding at least one chimeric nuclease which comprises a DNAbinding domain, a nuclease and a mitochondria or chloroplastlocalization signal and a nucleic acid expression construct whichcomprises at least one heterologous nucleic acid sequence which canupregulate the structural or functional gene of a mitochondria orchloroplast when targeted into the genome of the mitochondria orchloroplast, wherein the DNA binding domain mediates targeting of theheterologous nucleic acid sequence to the genome of the mitochondria orchloroplast, thereby generating male sterility in the plant.

Thus for example, the nucleic acid construct comprises a coding (e.g.,for a CMS associated gene) or non-coding (e.g., powerful promoter forenhancing expression of a CMS associated gene) heterologous nucleic acidsequence as well as a binding site for the chimeric nuclease (identicalto that on the mitochondria or chloroplast genome). Upon cleavage by thechimeric nuclease, the heterologous nucleic acid sequence is insertedinto the predetermined site in the genome of the chloroplast ormitochondria.

As mentioned hereinabove, cytoplasmic male sterility (CMS) is associatedwith mitochondrial dysfunction. To this effect, the chimeric nucleasesare designed to comprise a mitochondria localization signal (asdescribed in detail hereinabove) and cleavage sites which are specificfor the mitochondrial genome. Specific genes which may be upregulatedinclude, but are not limited to, the Petunia pcf chimera that is locatedwith close proximity to nad3 and rps12, the Rice (Oryza sativa) sequencewhich is downstream of B-atp6 gene (i.e. orf79), the Maize T-urf13 andorf221, the Helianthus sp. orf239 downstream to atpA, the Brassica sp.orfs which are upstream to atp6 (e.g. orf139 orf224 or orf138 andorf158). It will be appreciated that in order to induce CMS, thesegenomic sequences are typically transcribed in the plant, thus theteachings of the present invention envision targeting these sequences(e.g. by adding coding sequences) or overexpression thereof using theabove described methods as to achieve CMS.

It will be appreciated that CMS phenotype, generated by theincompatibility between the nuclear and the mitochondrial genomes, isused as an important agronomical trait which prevents inbreeding andfavors hybrid production.

As mentioned hereinabove, induction of CMS can also be achieved byoverexpression of a chloroplast gene such as β-ketothiolase.Overexpression of β-ketothiolase via the chloroplast genome has beenpreviously shown to induce CMS [Ruiz at al (2005) Plant Physiol. 1381232-1246]. Thus, the present teachings also envision targetingchloroplast genes or overexpression thereof (e.g. β-ketothiolase) usingthe above described methods in order to achieve CMS.

The present invention further envisages a method of generating aherbicide resistant plant. The method comprising introducing into theplant at least one viral expression vector encoding at least onechimeric nuclease which comprises a DNA binding domain, a nuclease and achloroplast localization signal, wherein the DNA binding domain mediatestargeting of the nuclease to a gene conferring sensitivity toherbicides, thereby generating the herbicide resistant plant.

It will be appreciated that in the field of genetically modified plants,it is well desired to engineer plants which are resistant to herbicides.Furthermore, most of the herbicides target pathways that reside withinplastids (e.g. within the chloroplast). Thus to generate herbicideresistant plants, the chimeric nucleases are designed to comprise achloroplast localization signal (as described in detail hereinabove) andcleavage sites which are specific for the chloroplast genome. Specificgenes which may be targeted in the chloroplast genome include, but arenot limited to, the chloroplast gene psbA (which codes for thephotosynthetic quinone-binding membrane protein Q_(B), the target of theherbicide atrazine) and the gene for EPSP synthase (a nuclear gene,however, its overexpression or accumulation in the chloroplast enablesplant resistance to the herbicide glyphosate as it increases the rate oftranscription of EPSPs as well as by a reduced turnover of the enzyme).

Chimeric nucleases and expression constructs of the present teachingsmay, if desired, be presented in a pack or dispenser device or kit. Thepack may, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor use.

It is expected that during the life of a patent maturing from thisapplication many relevant viral vectors and chimeric nucleases will bedeveloped and the scope of these terms is intended to include all suchnew technologies a priori.

As used herein the term “about” refers to ±10%

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Generation of Viral Vectors and Zinc Finger Nucleases

Plant Material

Rooted plantlets of Petunia hybrida lines B1, P720, Burgundy and RoyalBlue (Danziger-“Dan” Flower Farm, Mishmar Hashiva, Israel) wereroutinely used for Agrobacterium tumefaciens infection for transientexpression of foreign genes.

Other plants used for inoculation experiments included Solanumpimpinellifolium La121 (Davis University Gene bank), Capsicum annuumindra1750 (S&G Syngeta global LTD.), Arabidopsis thaliana[Greenboim-Wainberg et al. (2005) Plant Cell, 17: 92-102], Artemisia sp.(Danziger-“Dan” Flower Farm, Mishmar Hashiva, Israel), Nicotianabenthamiana [Radian-Sade et al., Phytoparasitica (2000) 28:79-86],Spinacia oleracea and Beta vulgaris (Eden Seeds, Reut, Israel),Nicotiana tabacum CV SAMSUNG [Levy et al. (2004) Plant Physiol 135:1-6],Nicotiana tabacum CV XANTHI [Ovadis et al. (1999) In “PlantBiotechnology and In Vitro Biology”, Kluwer Academic Press, theNetherlands, pp. 189-192], Cucumis sativus (Eden Seeds, Reut, Israel),Solanum melongena (M. Ben-Sachar LTD, Tel-Aviv, Israel), Gossypiumhirsutum cv. Siv'on [Saranga et al (2004) Plant, Cell and Environment27, 263-277) Brassica napus [Nesi et al., C. R. Biologies 331 (2008)763-771], Zea mays Var. Royalty F1 (M. Ben-Sachar LTD, Tel-Aviv,Israel). All plants were grown in a greenhouse under 25° C./20° C.day/night temperatures and under a natural photoperiod.

Plasmid Construction

pTRV1 a pYL44 binary T-DNA vector carrying the entire sequence of cDNAcorresponding to TRV Ppk20 strain RNA1 (GenBank accession No. AF406990)and pTRV2 (GenBank accession No. AF406991) vectors were used [Liu et al.Plant J (2002) 30: 415-429]. pTRV2 containing GUS (pTRV2-Gus) weregenerated by cloning GUS from pBI101 (Clontech Laboratories) into MCS(XbaI-SacI sites) of pTRV2. pTRV2 containing PAP1 (pTRV2-Δ2b-Pap) weregenerated by cloning PAP1 from pCHFS-PAP1 [Borevitz et al., The PlantCell (2000) 12:2383-2393] into MSC (EcoRI-BamHI sites) of pTRV2-Δ2b.

Generating pTRV2 for transient expression of target genes was carriedout by removal of the 300 by of the RNA2 2b gene from the originalvector. For this, pTRV2 DNA was digested with PvuII and EcoRI and thedeleted fragment containing part of 2b was replaced with a PCR fragment.This fragment was generated by PCR using pTRV2 DNA as a template andprimers A and B (see Table 1, below). It was digested with PvuII andEcoRI prior to recloning. The resultant plasmid (pTRV2Δ2b) was identicalto the original pTRV2 but lacking the 2b sequence. The plasmid pTRV2without 2b but with GUS (pTRV2Δ2b-Gus) was generated in the same wayexcept that pTRV2-Gus was used as a recipient plasmid instead of pTRV2.

For generation of pTRV2 containing full length of TRV 2b (pTRV2-2b) aPCR fragment of 1.5 Kbp was generated with primers A and C (see Table 1,below) using as a template pK202b-GFP [Vellios et al., Virology (2002)300:118-124]. The amplified fragment included the 2b gene (from Ppk20strain GenBank accession No. Z36974) with 5′ and 3′ UTRs plussub-genomic-promoter (sg-P) of the coat protein (CP) from Pea EarlyBrowning Virus (PEBV) (GenBank accession no X78455). This sg-P was siteddownstream to 2b. For generation of pTRV2-2b containing GFP (GenBankaccession No.U62637) downstream to sg-P (pTRV2-2b-Gfp), the PvuII-EcoRIfragment was transferred from pK202b-GFP to pTRV2 digested with the sameenzymes.

For generation of pTRV2Δ2b containing sg-P with downstream GFP(pTRV2Δ2b-Gfp), a PCR fragment was prepared using primers F and G* (seeTable 1, below) and pK202b-GFP as a template. This fragment was thendigested with Sad and SmaI and cloned into the pTRV2Δ2b digested withthe same enzymes. * Of note, Primer G adds a silence mutation to Gfp inorder to eliminate a Sad site upstream to the termination codon.

A Gus gene with and without Ω [Broido et al., Physiologia Plantarum(1993) 88: 259-266] was cloned into the MCS of pTRV2Δ2b-Gfp, upstream tosg-P of PEBV to generate pTRV2Δ2b-ΩGus-Gfp and pTRV2Δ2b-Gus-Gfp,respectively. The GUS fragment was generated following digestion ofpTRV2-Gus by EcoRI and Sad. ΩGus fragment was generated by cloning GUSinto SalI-BamHI sites downstream to the Ω sequence of TMV (Broido etal., supra) into pDrive (Qiagen) and then digesting the resultantplasmid with XbaI-KpnI to release ΩGus and reconstruct it intopTRV2Δ2b-Gfp.

pTRV2Δ2b containing ΩGus (pTRV2Δ2b-ΩGus) was generated by cloning GUSinto SalI-BamHI sites downstream to the Ω sequence of TMV (Broido etal., supra) into a pBluescript SK+ (Stratagen) and then digesting theresultant plasmid with KpnI-SacI to release ΩGus and reconstruct it intopTRV2Δ2b.

A pTRV2Δ2b carrying the Tomato bushy stunt virus silencing suppressorp19 (pTRV2Δ2b-p19) was constructed by transferring a 519-bp PCR fragmentencoding p19 (using primers D and E, see Table 1, below) from pCB301-p19[Voinnet et al., Plant J. (2003) 33: 949-956] into pTRV2Δ2b (Obermeieret al., Phytopathology (2001) 91:797-806].

All newly formed pTRV2 constructs were first transformed into E. coliand into Agrobacterium tumefaciens AGLO [Zuker et al Mol. Breeding(1999) 5:367-375]. Gus activity in Agrobacterium was evaluatedqualitatively with X-gluc solution as previously described [Zuker etal., supra]. GFP expression in Agrobacterium was analyzed qualitativelyusing fluorescence stereomicroscope (Leica Microsystems, Wetzlar,Germany).

pTRV2Δ2b containing sg-P with downstream DsRed2 (DsRed, GenBankaccession no AY818373 nucleotides 1395-2074 of DsRed, SEQ ID NO: 129,pTRV-Δ2b-sgP-DsRed) was generated by preparing a PCR fragment using as atemplate pSAT6-DsRed2-N1 and primers H and I (see Table 1, below). ThePCR product was then digested with HpaI and SacI, blunt ended with T4DNA polymerase and cloned (instead of GFP) into the pTRV-2A2b-GFPdigested with HpaI and SmaI.

Two different combinations of two fluorescence genes in pTRV2 wereconstructed:

A 2A-like 54 nucleotide sequence (GenBank accession no AF062037nucleotides 502-555, SEQ ID NO: 81), a Thosea asigna virus (TaV-T2A)[Donnelly et al (2001) J. Gen. Virol, 82, 1027-1041; Osborn et al (2005)Mol. Therapy, 12, 569-574], was utilized to create a bicistronic plasmidvector encoding a single, long, ORF consisting of the DsRed2 gene andthe NLS-EGFP genes. The sequence (coding for an 18 amino acid peptide),when inserted into single RNA molecule containing two ORFs, allowedseparate translation for the two ORFs [Donnelly et al (2001) supra;Osborn et al (2005), supra].

The T2A sequence was modified at the nucleotide level, based on Petuniacodon usage(httpllworldwidewebdotkazusadotordotjp/codon/cgi-bin/showcodon.cgi?species=4102)and the modified sequence was termed pTRV-T2A (see FIG. 2A).

A plasmid containing the pTRV-T2A sequence inserted between DsRed2 andNLS-EGFP was generated by first generating a PCR fragment usingpSAT6-NLS-P1-36-ZFN1 as a template following triple PCR reaction withtwo foreword primers J & K (Table 1, below) and reverse primer S (Table1, below) for FoKI. The resultant product was cloned into BamHI and Sadsites of pBluescript SK (pBS) lacking XhoI site, to generatepBS-T2A-P36-ZFN2. EGFP (GenBank accession no AY818363, SEQ ID NO: 130)was PCR amplified using primers N & O (Table 1, below) and cloneddownstream to NLS into XhoI and Sad digested pBS-T2A-P36-ZFN1, insteadof ZFN. The resultant plasmid was termed pBS-T2A-NLS-EGFP. The DsRed2was amplified using primers H & M (Table 1, below). The resultant stopcodon-lacking DsRed2 was ligated SalI-BamHI fragment into the pBST2A-NLS-EGFP upstream to T2A, yielding pBS-DsRed-T2A-NLS-EGFP with thebicistronic ORF. The DsRed-T2A-NLS-EGFP fragment from the plasmid wasthen ligated into HpaI-SacI sites of pTRV2-Δ2b-sgP to generatepTRV2-Δ2b-sgP-DsRed-T2A-NLS-EGFP.

A pTRV2-Δ2b-sgP containing two fluorescent marker genes was generatedunder separate subgenomic promoters in which pTRV2-Δ2b-sgP-GFP was firstdigested with SmaI. The PCR fragment generated using pTRV2-Δ2b-sgP-DsRedas a template and primers P & Q (see Table 1, below) was cloned intothis SmaI, to produce pTRV2-Δ2b-sgP-GFP-sgP-DsRed.

TABLE 1 Primers Primer Sequence A TGGAGTTGAAGAGTTATTACCGAACG(SEQ ID NO: 1) B AAGAATT CGAAACTCAAATGCTACCAA (SEQ ID NO: 2)C (PEBV-sgP R) TAGAATTCTCGTTAACTCGGGTAAGTGA (SEQ ID NO: 3) D (P19-F)AAACTCGAGATGGAACGAGCTATACAAGG AA (SEQ ID NO: 4) E (P19-R)AAACCCGGGAGAGTCTGTCTTACTCGCCTT CT (SEQ ID NO: 5) F (5′-PEBV-sgP-F)AAGAGCTCGAGCATCTTGTTCTGGGGTT (SEQ ID NO: 6) G (GFPuv-3′-SmaI)ACCCGGGTTATTTGTAGAGTTCATCCATGC CA (SEQ ID NO: 7) H (DsRFP-F-HpaI)AGTTAACGAGATGGCCTCCTCCGAGA (SEQ ID NO: 53) I (DsRed2-R-SacI)TAGAGCTCTCACAGGAACAGGTGGTGGC (SEQ ID NO: 54) J (T2A-F-BamHI)TTTGGATCCGAAGGAAGAGGATCTCTTCTT ACTTGTGGTGATGTTGAAGAG (SEQ ID NO: 55)K (T2A-NLS-F- TTACTTGTGGTGATGTTGAAGAGAATCCTG first) GACCAAAAAAGAAGAGAAAG (SEQ ID NO: 56) L (R-FokI SacI)AAGAGCTCTTAGGATCCAAAGTTTATCTC (SEQ ID NO: 57) M (DsRFP-R-AGGATCCCAGGAACAGGTGGTGGC BamHI) (SEQ ID NO: 58) N (EGFP-F-XhoI)A TCT CGA GTG AGC AAG GGC GA (SEQ ID NO: 59) O (EGFP-R-SacI)AGAGCTCTACTTGTACAGCTCGTCCATG (SEQ ID NO: 60) NLS (uppercase)atggtgCCAAAAAAGAAGAGAAAGGTAGAA GACCCCtctcgag (SEQ ID NO: 61)P (2sg F1 Sma) CCCGGGATTTAAGGACGTGAACTCTGT (SEQ ID NO: 62)Q (dsRed R678 Sma) CCCGGGTCACAGGAACAGGTGGT (SEQ ID NO: 63)R (NLS linking AGTTAACGAGATGCCAAAAAAGAAGAGAAA gene) GGT (SEQ ID NO: 64)S (FokI-m-R-SacI) AAGAGCTCTTAaGATCCAAAGTTTATCTC (SEQ ID NO: 65)T (FokI-m-R-SmaI) ACCCGGGTTATCCAAAGTTTATCTCGCCGT (SEQ ID NO: 66)U (F-QEQ-ZFN) AACTCGAGAAAAACTGCGGAACGGA (SEQ ID NO: 67)V (Rssu tp-F-HpaI) AGTTAACGAGATGGCTTCTATGATATCCT CT (SEQ ID NO: 68)W (ATP-β-tp-F-HpaI) AGTTAACGAGATGGCTTCTCGGAGG (SEQ ID NO: 69)

Cloning of pTRV2 Viral Vectors Allowing Targeting of Gene Products toPlastids

To generate EGFP targeted to chloroplast, inventors amplified a PCRfragment containing transit peptide of Pea ribulose-1,5-bisphosphatecarboxylase small subunit (Rssu) (GenBank accession no X00806,nucleotides 1086-1259, SEQ ID NO: 138) fused to EGFP with primers V & O(see Table 1, above) using the plasmid pTEX-Rssu-GFP previouslydescribed by Bezawork [Bezawork (2007) M.Sc Thesis, submitted toAgricultural Research Organization, Volcani center and the Faculty ofAgriculture] as a template. The PCR product, following blunting withHpaI, was cloned downstream to sgP into pTRV2-Δ2b-sgP to producepTRV2-Δ2b-sgP-Rssu-EGFP.

To generate EGFP targeted to mitochondria, inventors amplified a PCRfragment containing a signal peptide of Nicotiana sylvestris ATPase betasubunit (ATP-(3) (GenBank accession no U96496, nsatp2.1.1, nucleotides12 to 167, SEQ ID NO: 139) fused to EGFP with primers W & O (see Table1, above) using the plasmid pTEX-ATP13-GFP previously described byBezawork [Bezawork (2007), supra] as a template. The PCR product,following blunting with HpaI, was cloned downstream to sgP intopTRV2-Δ2b-sgP to produce pTRV2-Δ2b-sgP-ATPβ-EGFP.

Inoculation of Plants with TRV Vectors

Agrobacterium tumefaciens (strain AGLO) transformed with pTRV1, pTRV2and pTRV2 derivatives were prepared as previously described [Liu et al.,Plant J (2002) 30: 415-429]. The Agrobacterium culture was grownovernight at 28° C. in LB medium complemented with 50 mg/L kanamycin and200 μM acetosyringone (A.S.). The cells were harvested and resuspendedin inoculation buffer containing 10 mM MES, 200 μM A.S. and 10 mM MgCl2to an OD600 of 10. Following an additional 3 hours of incubation at 28°C., the bacteria with the pTRV1 was mixed with the bacteria containingthe pTRV2 derivates at a 1:1 ratio. When co-infection of more then onepTRV2 was involved, the Agrobacteria with pTRV1 were always 50% in themixture. 200-400 μl of the Agrobacteria mixture was used for injectioninto the stem. Agrobacteria were also injected into the exposed shootsurface following removal of the apical meristems.

Another option for infection was leaf infiltration using a syringewithout a needle: Agrobacteria content of the syringe was dischargedinto the scratched surface of the leaf. For infection of plants withTRV, without the use of Agrobacteria, first N. benthamiana or N.clevelandii (the usual host for TRV) was inoculated with pTRV1 and pTRV2or its derivatives. About 15 to 21 days post infection (dpi) plantleaves (as a source for freshly prepared sap infection) were collectedand the sap was extracted in 20 mM phosphate buffer (pH-6.8) by mortarand pestle.

For virion infection of plants (TRV infection without use ofagrobacteria), inventors first inoculated Petunia hybrida, Nicotianatabacum cv Samsung or N. benthamiana, (the usual hosts for TRV) withpTRV1 and pTRV2 (or its derivatives). About 5 to 21 days post infection(dpi) inventors collected plant leaves and extracted the sap in 20 mMphosphate buffer pH=6.8 and a surfactant (e.g. 0-0.03% Silwet L-77) bymortar and pestle. The TRV containing sap was dripped onto cheeseclothor centrifuged and following addition of carborundum fine powder (toimprove infection) stems and leaves of young (approximately 1 month old)plants were gently scratched.

Sap infection of in-vitro grown plants: sap was first passed through0.22 μm filter and then stems of tissue culture propagated plants wereinjured and infected using syringe and needle.

For Zea mays (monocotyledons) infection, the seeds were incubated withthe sap during swelling and sprouting (for approximately 1-2 weeks).

In addition to AGLO strain of Agrobacterium, inventors also successfullyused the EHA-105 [Tovkach et al., Plant J. (2009) 57, 747-757] strainfor the delivery of various pTRV constructs.

For inoculation of in vitro grown plants using A. tumefaciens AGLO orEHA-105 bacteria, a MS solution [Murashige and Skoog, Physiol Plant(1962) 15:473-497] without glucose but with 10 mM MgSO₄ and 50 μg/mlacetosryngone (A.S.) was used and the concentration of the bacteria wasreduced to 0.08-0.8 OD 600 nm for each TRV. Infection was performedessentially as above with sap or via vacuum infiltration.

Expression Analysis

Each expression experiment was repeated three times and each experimentincluded at least four plants per treatment. Tested plant meristems (atleast 2 per plant) were collected several times during the experimentalcourse. GFP imaging was completed using UV illumination and photographswere taken using fluorescence stereomicroscope (Leica Microsystems,Wetzlar, Germany) equipped with a digital camera and a filter set forexcitation at 455-490 nm and emission at more than 515. Gus activity wasevaluated using the substrate 1 mM 5-bromo-4chloro-3-indolyl-β-D-glucoronic acid (X-gluc., Duchefa Biochemie B.V.Haarlem, Netherlands) in an appropriate buffer (Zuker et al., supra).Prior to an overnight incubation with the substrate mixture at 37° C.,plant tissue was vacuum infiltrated with the substrate for 30 minutes.The substrate solution was then exchanges with 75-95% ethanol for a fewdays for chlorophyll bleaching and the tissue was observed using astereomicroscope.

DsRed2 imaging was completed using UV illumination and photographs weretaken using fluorescence stereomicroscope (Leica Microsystems, Wetzlar,Germany) equipped with a digital camera and a filter set for excitationat 530-560 nm and emission at 590-650.

EGFP and DsRed2 imaging was also generated using a confocallaser-scanning microscope (CLSM510, Zeiss Jena Germany). For EGFP,excitation was set at 488 nm and emission at 505-530 nm, for DsRED2,excitation was set at 545 nm and emission at 585-615 nm.Autofluorescence of chlorophyll, excitation was set at 488 nm andemission at more than 650 nm.

Preparation of Protoplasts

Petunia leaves were used to generate protoplasts as previously describedby Locatelli [Locatelli et al, Plant Cell Reports (2003) 21: 865-871].

Transgenic Plants

The binary vector pRCS2[QQR-TS*::GUS] previously described by Tovkach[Tovkach et. al. (2009), supra] was transferred to Agrobacteriumtumefaciens which was then used to transform Petunia hybrida cv Burgundyand Nicotiana tobaccum cv. Samsung using the standard leaf disctransformation method [Guterman et al., Plant Mol. Biol. (2006)60:555-563].

Identification of Non-Coding Genomic Sequences of Petunia

A genomic DNA of Petunia cv. Royal Blue was prepared using a standardprotocol. Initial digestion of the genomic DNA with EcoRI and HindIIIwas carried out followed by agarose (1%) gel electrophoresis. Next,1-1.5 Kbp fragments were extracted from the gel by a gel extraction kit(iNtRON Biotechnology, INC. LTD, KOREA). These fragments were ligated topBS-SK (IRA Company) to form a semi-genomic library in E. coli.Sequences of 110 genomic fragments were generated by Macrogen Inc.(Seoul, Korea). Two BLAST analyses (nucleotide blast and tblastx) wereperformed with the generated genomic petunia sequences againstnucleotide collection and non-human, non-mouse ESTs libraries, to allowelimination of all the putatively transcripted/translated DNAs. Allsequences with a BLAST E value higher then 5 were further evaluated toidentify those with the shortest ORF, for all six reading frames, andwith minimum repetitive AAAA and TTTT regions. Finally two Petuniagenomic DNA fragments were selected as non-coding, non repetitivesequences, P1-25 (1.2 Kbp, FIG. 2B) and P1-36 (1.175 Kbp, FIG. 3).

Within these sequences a target site for zinc finger nuclease (ZFN) wasdesigned. Zinc finger proteins are capable of recognizing virtually any18 by long target sequence, enough to specify a unique address withinplant genome. The target sites (artificial-palindrome-like sequencetargets, marked in blue in FIGS. 2-3) used were:

P1-25 site 1:  TCC-TCC-TGC (SEQ ID NO: 10) site 2: GAG-GGG-GAA(SEQ ID NO: 11) P1-36 site 1: ACC-ACC-ATC (SEQ ID NO: 12) site 2:GGT-TGA-GAG (SEQ ID NO: 13)

Identification of PDS and FHT Sequences as ZFN Target Sites in Petunia

The sequence of phytoene desaturase (PDS) exon from Petunia hybrida RBwas confirmed by resequencing (based on GenBank accession no AY593974.1,SEQ ID NO: 131) and utilized as target sites for ZFNs. The highlightedsequences (FIG. 28A) were utilized as the target sites of PDS-ZFNproteins (SEQ ID NOs: 71 and 73).

The sequence of flavanone 3 beta-hydroxylase (FHT) exon from Petuniahybrida RB (GenBank accession no AF022142.1, SEQ ID NO: 133) wasidentified and utilized as target sites for ZFNs. The sequence wasconfirmed by resequencing. The highlighted sequences (FIG. 29) wereutilized as the target sites of FHT-ZFN proteins (SEQ ID NOs: 75 and77).

Design of Zinc Finger Nucleases (ZFNs)

The zinc finger proteins coding regions were designed based on azinc-finger-framework consensus sequence formerly developed byDesjarlais and Berg [Desjarlais and Berg, Proc. Natl. Acad. Sci. USA(1993) 90: 2256-2260]. For example, expression of zinc fingerendonuclease with the expected affinity to the gagggggaa sequence onP1-25 Petunia random DNA fragment (site 2, SEQ ID NO: 11) the zincfinger 262 by domain was assembled by PFU polymerase (Invitrogen) in aPCR reaction from the set of the following overlapping oligos: BBO1(5′GAAAAACCTTACAAGTGTCCTGAATGTGGAAAGTCTTTTTCT, SEQ ID NO: 14), BBO2M(5′CAGCGAACACACACAGGTGAGAAGCCATATAAATGCCCAGAATGTGGTA AATCATTCAG, SEQ IDNO: 15), BBO3M (5′CAACGGACCCACACCGGGGAGAAGCCATTTAAATGCCCTGAGTGCGGGAAGAGTTTTT, SEQ ID NO: 16), FtsH2-Z1.1-GAA (SEQ ID NO: 17), P1-25-ZFN2.2GGG (SEQ ID NO: 18) and P1-25-ZFN2.3 GAG (SEQ ID NO: 19) followed by PCRamplification using the BBO1-XhoI-F (SEQ ID NO: 20) and SDO3-Spe1-R (SEQID NO: 21) primers producing the DNA binding domain P1-25-ZFN2bd.

In each PCR reaction, the BBOs and SDOs were mixed at 0.005 μMconcentration and amplified for 35 cycles with PFU polymerase(Invitrogen). Similar strategies, only using different oligos (see Table2, below) have been employed for the assembly of the P1-25-ZFN1bd,P1-36-ZFN1bd and P1-36-ZFN2bd DNA binding domains. An outline of the PCRprocedure for assembly of ZF binding domains used in this work isillustrated in FIG. 4.

TABLE 2 Sequences of overlapping oligos used for genera-tion of DNA binding domains P1-25-ZFN1bd,  P1-36-ZFN1bd and P1-36-ZFN2bdP1-25-ZFN1bd 41 P1-25-ZFN1.1 ACCTGTGTGTGTTCGCTGGTGACGTTCAAGATG GGAAGCACGCTGAGAAAAAGACTTTCCACA (SEQ ID NO: 22) 42 P1-25-ZFN1.2CCCGGTGTGGGTCCGTTGGTGACGTTCAAGAT GGA GAGCACGCTGACTGAATGATTTACCACA(SEQ ID NO: 23) 24 ZFN-IV-Mod TCCAGTATGAGTACGTTGATGACGACGCAAA SDO3 GCATCTCCAGACTGTGAAAAACTCTTCCCGCAC (SEQ ID NO: 24) P1-25-ZFN2bd 25FtsH2-Z1.1- ACCTGTGTGTGTTCGCTGGTGCTTCTGA GAAAGGTTGCTAGACTGAGAAAAAGACTTTCCACA (SEQ ID NO: 17) 43 P1-25-ZFN2.2CCCGGTGTGGGTCCGTTGGTGACGA GGG ACCAACTTATCAGAACGACTGAATGATTTACCA CA(SEQ ID NO: 18) 44 P1-25-ZFN2.3 TCCAGTATGAGTACGTTGATGACGAACCAA GAGATTATCAGAACGTGAAAAACTCTTCCCGCAC (SEQ ID NO: 19) P1-36-ZFN1bd 45P1-36-ZFN1.1 ACCTGTGTGTGTTCGCTGGTGACGAA GGTCAAGATGTCCAGAAGTAGAAAAAGACTTTCCA CA (SEQ ID NO: 25) 29 FtsH2-Z3.2-CCCGGTGTGGGTCCGTTGGTGACGAACAAG GGT ATGTCCAGAAGTACTGAATGATTTACCACA(SEQ ID NO: 26) 35 FtsH2-Z1a.3 TCCAGTATGAGTACGTTGATGACGAACCAAA GATTTTCCAGAAGTTGAAAAACTCTTCCCGCAC (SEQ ID NO: 27) P1-36-ZFN2bd 46P1-36-ZFN2.1  ACCTGTGTGTGTTCGCTGGTGACGAACCAAA GAGTTATCAGAACGAGAAAAAGACTTTCCACA (SEQ ID NO: 28) 47 P1-36-ZFN2.2CCCGGTGTGGGTCCGTTGGTGAGAAGCCAAAT TGA GTCCAGCCTGACTGAATGATTTACCACA(SEQ ID NO: 29) 48 P1-36-ZFN2.3 TCCAGTATGAGTACGTTGATGACGAACAAGAT GGTGTCCAGAAGTTGAAAAACTCTTCCCGCAC (SEQ ID NO: 30)

Amplified DNA binding domains were cloned as an XhoI-SpeI fragment intothe same sites of pSAT6-NLS-FokI, producing the pSAT6-NLS-P1-25-ZFN1(SEQ ID NO: 31), pSAT6-NLS-P1-25-ZFN2 (SEQ ID NO: 32),pSAT6-NLS-P1-36-ZFN1 (SEQ ID NO: 33) and pSAT6-NLS-P1-36-ZFN2 (SEQ IDNO: 34) expression vectors (FIGS. 5A-D). pSAT6-NLS-FokI consists of pSATvector [GeneBank AY818383, Tzfira et al., Plant Mol Biol (2005) 57:503-516], 30 by of the NLS (nuclear localization signal, SEQ ID NO: 46)cloned into NcoI-XhoI sites and a 584 by fragment of FokI endonuclease(nucleotides 1164 to 1748; GeneBank J04623) cloned into SpeI-BamHI sitesof pSAT6.

For expression of His tagged zinc finger endonucleases in E. coli cells,the NLS-P1-25-ZFN1, NLS-P1-25-ZFN2, NLS-P1-36-ZFN1 and NLS-P1-36-ZFN2fragments were cloned as NcoI-BamHI inserts from their correspondingplasmids into the same sites of a modified pET28 (pET28.5X, FIG. 5E),producing pET28.5X-NLS-P1-36-ZFN1, pET28.5X-NLS-P1-36-ZFN2,pET28.5X-NLS-P1-25-ZFN1 and pET28.5X-NLS-P1-25-ZFN2. In pET28.5X,assembled ZFNs are cloned downstream of T7 promoter of pET28 vector(Novagen). Complete ZFN constructs also contained a sequence coding for6×His-tag at the C terminus of the protein.

Expression of pET28.5X-NLS-P1-36-ZFN1, pET28.5X-NLS-P1-36-ZFN2,pET28.5X-NLS-P1-25-ZFN1 and pET28.5X-NLS-P1-25-ZFN2 was performed inBL21 GOLD (DE3) PlyS cells (Stratagene). Cell cultures were grown in 100ml LB medium complemented with Kan (50 ug/ml) and 100 μM ZnCl₂ at 22° C.until OD₆₀₀ was 0.6 and then cells were induced with 0.7 mM IPTG for 3hours. Cells were harvested by centrifugation, resuspended in 35 mlmixture containing 25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol and100 μM ZnCl₂, and lysed twice through a French Press. The protein wasloaded on 0.5 ml Ni-NTA agarose beads (Qiagen) and eluted with 1 mlbuffer containing 500 mM imidazole. Eluted protein was stored at −20° C.in 50% glycerol.

Various quantities of E. coli and Ni-NTA purified ZFNs were mixed with0.5 μg of target DNA for in vitro digestion using NEB4 buffer anddigested DNA substrates were separated by agarose gel electrophoresis.

Cloning of pTRV2 Viral Vectors Allowing Expression of ZFNs

ZFN inserts in pSAT constructs (pSAT6-P1-36-ZFN1, pSAT6-P1-36-ZFN2,pSAT6-PDS-ZFN1, pSAT6-PDS-ZFN2, pSAT6-FHT-ZFN1, pSAT6-FHT-ZFN2) startand end with nonspecific domains, NLS and FokI respectively. To generatepTRV2 suitable for delivery and expression of different ZFNs in plantcells, inventors amplified ZFNs using the forward primer R and thereverse primers L (for pSAT6-P1-36-ZFN2, pSAT6-PDS-ZFN1, pSAT6-FHT-ZFN1)or T (for pSAT6-P1-36-ZFN1, pSAT6-PDS-ZFN2, pSAT6-FHT-ZFN2) (see Table1, above). The resultant amplification products were digested with HpaIand Sad (for primer L) or SmaI (for primer T), blunt ended (in case ofSad) and inserted into HpaI-SmaI sites of pTRV2-Δ2b-sgP. This lead tothe generation of pTRV2-Δ2b-sgP-PDS-ZFN1 (FIG. 31A, SEQ ID NO: 70),pTRV2-Δ2b-sgP-PDS-ZFN2 (FIG. 31B, SEQ ID NO: 72); pTRV2-Δ2b-sgP-FHT-ZFN1(FIG. 32A, SEQ ID NO: 74), pTRV2-Δ2b-sgP-FHT-ZFN2 (FIG. 32B, SEQ ID NO:76); pTRV2-Δ2b-sgP-P36-ZFN1 (SEQ ID NO: 33) and pTRV2-Δ2b-sgP-P36-ZFN2(SEQ ID NO: 34).

To generate pTRV2-Δ2b-sgP-QEQ-ZFN (SEQ ID NO: 82), inventors firstgenerated a PCR product using primers U and S (see Table 1, above) andpSAT4.hspP.QQR [Tovkach et al (2009), supra] as a template. The productwas digested with XhoI and SpeI and inserted into XhoI and SpeIdigested, ZF lacking, plasmid pTRV2-Δ2b-sgP-P36-ZFN2 (FIG. 33).

To generate a construct containing two ZFNs linked with T2A(pTRV2-Δ2b-sgP-P36-ZFN2-T2A-P36-ZFN1, SEQ ID NO: 84), the BamHI fragmentfrom pTRV2-Δ2b-sgP-P36-ZFN2 containing the sgP-NLS-P1-36-ZFN2 was clonedinto the unique BamHI site in pBS-T2A-NLS-36-ZFN1. This generatedpBS-sgP-P36-ZFN2-T2A-P36-ZFN1. This plasmid was digested with Sad andthe released fragment containing sgP-P36-ZFN2-T2A-P36-ZFN1 was clonedinto pTRV2-Δ2b linearized with Sad to generatepTRV2-Δ2b-sgP-P36-ZFN2-T2A-P36-ZFN1.

To generate pTRV2-Δ2b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 (SEQ ID NO: 86) andpTRV2-Δ2b-sgP-FHT-ZFN1-T2A-FHT-ZFN2 (SEQ ID NO: 88), inventors firstdigested pBS-T2A-NLS-36-ZFN1, pSAT6-FHT-ZFN2 and pSAT6-PDS-ZFN2 withXhoI & SpeI (flanking ZF sequences). Inventors then ligated fragmentsreleased from the latter two plasmids into digested pBS-T2A-NLS-36-ZFN1to create pBS-T2A-NLS-PDS-ZFN2 and pBS-T2A-NLS-FHT-ZFN2. PlasmidspTRV2-Δ2b-sgP-PDS-ZFN1 and pTRV2-Δ2b-sgP-FHT-ZFN1 were digested withBamHI to release sgP-PDS-ZFN1 and sgP-FHT-ZFN1, which were then ligatedinto BamHI digested pBS-T2A-NLS-PDS-ZFN2 and pBS-T2A-NLS-FHT-ZFN2,respectively. These plasmids were digested with Sad and the releasedfragments were cloned into unique Sad site of the MCS in pTRV2-Δ2byielding pTRV2-Δ2b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 andpTRV2-Δ2b-sgP-FHT-ZFN1-T2A-FHT-ZFN2, respectively.

Example 2 Expression of Foreign Genes by pTRV2-Δ2b Vectors

The RNA2-2b fragment (300 by of SEQ ID NO: 43 depicted in SEQ ID NO: 50)was entirely removed from pTRV2 (GenBank accession No. AF406991) togenerate the pTRV2-Δ2b (see Example 1 hereinabove and FIGS. 6A-B).Inventors were interested in the expression of the reporter genes in themeristematic tissues and hence reporter gene expression was evaluated inthese tissues. Petunia plants were inoculated with pTRV2-Δ2b-GUS andpTRV2-GUS and the efficiency of foreign gene expression (i.e. GUS) bythese vectors was compared. GUS expression was evaluated a week to twomonth following infection and percent of meristems expressing GUS out oftotal number of analyzed meristems was presented (see Table 3, below).As clear from the results, pTRV2 without the 2b region was much moreefficient in GUS expression in meristimatic tissues. Furthermore, no GUSstaining was noticeable in petunia plants inoculated with TRV lackingGUS. Typical GUS expression in the meristems of inoculated plants isillustrated in FIGS. 7A-E. As clear from FIG. 7E, GUS staining inpetunia plants following inoculation with pTRV2-Δ2b-GUS was noticeableeven after numerous rounds of propagation (via axillary meristems) intissue culture. Additional marker genes used to assay applicability ofpTRV2 based vector for expression of foreign genes were GFP and PAP1.Inoculation of petunia and other plants (Capsicum annuum, Solanumpimpinellifolium, Nicotiana benthamiana, Arabidopsis thaliana, Artemisiaannua, Spinacia olerace and Beta vulgaris) with these vectors led toexpression of the reporter genes in meristems of all analyzed plants(FIGS. 8A-G).

TABLE 3 Expression of GUS in Petunia plants inoculated with pTRV2-Δ2b-GUS and pTRV2-GUS Days post inoculation 7 13 21 37 48 pTRV2-GUS 50% 40%11% 7% 0% TRV2-Δ2b-GUS 45% 60% 54% 40% 60%

Example 3 Enhancement of Foreign Gene Expression by Ω TranslationalEnhancer

The 70 by at the 5′UTR of TMV (Ω) is a non-coding sequence shown to be atranslational enhancer (SEQ ID NO: 44) [Gallie et al., Nucl. Acid. Res.(1987) 15:8693-8710]. Ω was cloned upstream to the reporter gene in thepTRV2 viral vectors in order to evaluate whether it can promoteexpression levels of foreign genes (see FIGS. 6A, C). As illustrated inFIGS. 9A-B, inoculation of petunia plants with pTRV2-Δ2b-ΩGUS vectorsresulted in higher GUS activity levels as compared to that obtainedusing pTRV2-Δ2b-GUS vectors (lacking the Ω enhancer). It should be notedthat the Ω fragment did not affect the percent of meristems expressingGUS out of total number of analyzed meristems.

Example 4 Co-Expression of Two Foreign Genes by pTRV2 Vectors

Two approaches were developed to allow co-expression of two codingsequences. First, a pTRV2 vector was generated that carries anadditional subgenomic promoter (sgP) sequence, hence allowingco-expression of two coding sequences (see FIGS. 6A, D). Coat proteinsubgenomic promoter of PEBV was used to this end. To test vectoractivity, GFP reporter gene was cloned downstream to this subgenomicpromoter to create pTRV2-Δ2b-sgP-GFP. Inoculation of N. benthamianaplants with this vector, led to expression of GFP in meristematictissues (FIG. 10A).

In an alternative approach aiming to co-express two foreign genes, N.benthamiana plants were co-inoculated with pTRV2-Δ2b-GUS andpTRV2-Δ2b-GFP (FIGS. 10B-C). Co-expression of both reporter genes wasrevealed based on the analyses of GFP expression in the tissue followedby GUS staining of the same tissues.

Example 5 Expression of Foreign Genes by pTRV Vectors in a Wide Varietyof Plants

Inoculation of different plants (e.g. N. benthamiana, N. tobaccum andPetunia hybrida) with pTRV1 and pTRV2-Δ2b-sgP-DsRed lead to a highexpression level of the marker gene in cells of these plants (FIGS.11A-F). Continuous (several months) strong expression, due to systemicinfection, was easily detected in different parts of these plants (FIGS.12A-H). Inoculation of various plants (e.g. Cucumis sativus, Solanummelongena, Gossypium hirsutum cv. Siv'on (cotton), Brassica napus(canola), Beta vulgaris (beet), Spinacia oleracea) with this vector leadto expression of the reporter genes in all analyzed plants (FIGS.13A-K).

Example 6 Expression of Foreign Genes by TRV Vectors in Monocots

To assay the applicability of pTRV2 based vectors for expression offoreign genes in monocots (e.g. maize), seeds were incubated with sapgenerated from petunia plants infected with pTRV1 andpTRV2-Δ2b-sgP-DsRed (as depicted in detail in Example 1, hereinabove).FIGS. 14A-C show clear expression of DsRed in coleoptile.

Example 7 Mitochondrial & Chloroplast Plastids TRV Vector-MediatedPlastid-Targeted Expression

As described in Example 1, hereinabove, inventors have constructed twovectors pTRV2-Δ2b-sgP-Rssu-EGFP and pTRV2-Δ2b-sgP-ATPβ-EGFP containing achloroplast transit peptide and a mitochondrial signal peptide,respectively. Inventors agroinfiltrated both pTRV2-Δ2b-2sgP-tp-EGFP (tphere is stand for transit peptide and signal peptide) into Petuniahybrida cv RB and N. benthamiana. The EGFP expression was first analyzedby fluorescent stereomicroscope, then the fluorescent leaf zones wereanalyzed by confocal laser scanning microscope. The chloroplast size andauto fluorescence (excitation at 488 nm, emission more than 650 nm)enabled to localize the expression of EGFP (excitation at 488 nm,emission 505-530 nm) to the chloroplast (FIGS. 15A-G).

For mitochondrial identification, protoplasts were prepared and redfluorescent mitochondrial specific reagent (MitoTracker® Invitogen inc.USA) was employed. The use of excitation 545 nm and emission 585-615 nmallowed distinguishing the fluorescence of chloroplasts from that ofMitoTracker. According to the size and location and mitotracker signal,inventors revealed that the expression of EGFP was localized tomitochondria (FIGS. 16A-K).

Example 8 Co-Expression of Two Reporter Genes in Various Plants

Several approaches were used to simultaneously express two genes inplant (Petunia hybrida, N. benthamiana or N. tabacum) cells. In oneapproach, plants were inoculated simultaneously with two TRV vectors,one carrying one marker gene and another carrying another marker gene.Specifically, plants were co-infection with pTRV1 and two pTRV2 vectors,pTRV2-Δ2b-sgP-DsRed and pTRV2-Δ2b-sgP-Rssu-EGFP. Results of confocalfluorescent scanning microscopy of in vitro Agroinfiltrated Nicotianatabacum cv Xanthi plants showed co-expression of both EGFP and DsRed(FIGS. 17A-D).

The second and third approaches for co-expression of two genes weredemonstrated using pTRV2 constructed with two reporter genes in tandem.The genes were either separated by T2A (FIGS. 18A-L) or were driven byseparate double subgenomic promoters (FIGS. 19A-J). As depicted in FIGS.18A-L, the co-expression of GFP and DsRed was clear followinginoculation of plants with pTRV2-Δ2b-sgP-DsRed-T2A-NLS-EGFP. Similarly,as depicted in FIGS. 19A-J, the co-expression of GFP and DsRed was clearfollowing inoculation of plants with pTRV2-Δ2b-sgP-GFP-sgP-DsRed.

Example 9 Generation of Specific Zinc Finger Nucleases (ZFNs)

Petunia non repetitive putatively non-coding genomic sequences wereidentified following sequencing of 110 genomic fragments (see Example 1,hereinabove). Two sets of ZFN, 25-ZFN-1, 25-ZFN-2 and 36-ZFN-1,36-ZFN-2, were synthesized in order to form a double cut in thePetunia's specific DNA sequences, P1-25 and P1-36, respectively. To testnuclease activity of the generated ZFNs, PCR fragments were generatedcontaining target sequences in a palindrome-like form and thesefragments were incubated with the specific ZFNs. As illustrated in FIG.20, PCR fragments were digested by each ZFN to the expected sizes.

To further verify ZFNs activities, pBS vectors carrying P1-36 sequenceswere generated. Incubation of 740 by fragment of P1-36 carrying targetsequences (generated by digestion of pBS-P1-36 with NcoI/BamHI*) withpurified ZFNs, 36-ZFN1 and 36-ZFN2, yielded fragments of expected sizes(FIG. 21, depicted by arrows). Of note, BamHI as well as SmaI are partof the multiple cloning sites (MCS) of pBS, right upstream to thecloning site EcoRI. The NcoI site is part of the P1-36 sequence and 200by downstream to P1-36 site2. Furthermore, as expected, 36-ZFN1 and36-ZFN2 individually did not yield digestion products. Moreover, asillustrated in FIG. 22, the combination of 36-ZFN-1 and 36-ZFN-2successfully digests the target sequence P1-36 carried by pBS-P1-36.

Furthermore, Petunia phytoene desaturase (PDS) genomic sequences wereidentified following sequencing of genomic fragments (see FIG. 28A).Sets of ZFN, PDS-ZFN1 and PDS-ZFN2, were synthesized (as depicted indetail in Example 1, hereinabove) in order to form a double cut in thePetunia's specific PDS DNA sequences. To test nuclease activity of thegenerated ZFNs, plasmids were constructed to carry semi-palindromictarget sequences and these plasmids were incubated with the specificZFNs. As illustrated in FIG. 23, digestion of plasmids carryingartificial target sites PDS1 and PDS2 (PDS-TS1 and PDS-TS2,respectively) by specific ZFNs was carried out. Plasmids were digestedby AgeI and PDS-ZFN1 or PDS-ZFN2 to the expected sizes.

In conclusion, the results conclusively show that foreign genes can beexpressed in plants meristems, including petunia meristems.Additionally, these results show the specific digestion in vitro ofpetunia DNA by ZFNs.

Example 10 Generation of Viral Expression Vectors Comprising ZFNs

The expression of ZFNs by pTRV expression vectors is underway todetermine the best approach to co-express ZFNs in petunia. Threeapproaches are being tested each of which is first tested with twofluorescent reporter genes (GFP and DsRFP) as depicted in detail above.These fluorescent reporter genes are delivered to Petunia plants andtheir co-expression within meristematic cells are examined usingfluorescent microscopy. Based on these results, ZFNs expression vectorswill be generated.

In the first approach, each gene is cloned separately into thepTRV2-Δ2b-Ω into the SalI-SacI site. Plants are then co-infected withthe two plasmids of pTRV2 (one carrying GFP and the other carryingDsRFP) simultaneously.

In the second approach genes are both introduced into the same pTRV2(pTRV2-Δ2b-Ω), each gene with a different sub-genomic promoter (sg-P).Plants are then infected with the pTRV2 plasmid.

The third approach is based on a ‘2A like’ protein that is able tocleave itself at the C termini [(Donnelly et al., J. Gen. Virol. (2001)82: 1027-1041; Osborn et al., Molecu. Therapy (2005) 12: 569-574]. The‘2A-like’ protein sequence, EGRGSLLTCGDVEENPGP (SEQ ID NO: 41, T2A) ofinsect virus Thosea asigna mediates an efficient co-translationalcleavage event resulting in the release of each individual proteinproduct. The C-termini Pro is the only amino acid that remains with thedownstream protein following the self-cleavage. An oligomer wassynthesized that encodes these 18 amino acids of the ‘2A-like’ protein(T2A). The nucleotide sequence was designed based on the Petunia codonusage (Codon Usage Database http://wwwdotkazusadotordotjp/codon). Toco-translationally express GFP and DsRFP, they are cloned in frame intopTRV2-Δ2b-Ω separated by the T2A 18 amino acids. To deliver ZFNsproteins to the nucleus via this approach, nuclear localization signal(NLS) sequences that start with Pro, are fused to their 5′ ends. Hence,Pro are shared by NLS and T2A, i.e. the last 3′ Pro of the T2Arepresents the first amino acid of the NLS. This eliminated the need tointroduce additional foreign sequences 5′ to NLS. The final insert isKpnI-Ω-NLS-ZFN2-T2A-NLS-ZFN1-SacI. For example, usingpTRV2-Δ2b-sgP-CP-PEBV (FIGS. 24A-B) carrying DsRFP and GFP, separated byT2A, petunia tissues co-expressing both reporter genes were generated.

An optional modification to the third approach is to clone one of theZFNs with the T2A at the N termini (T2A-NLS-ZFN1 fragment) downstreamand in frame with TRV1 16 K gene (FIG. 6E). In this case the Agroinfection of Petunia plants is performed with modified pTRV1 and pTRV2,each carrying only one foreign ZFN gene.

Example 11 pTRV-Vector-Mediated Activation of GUS Expression in Plants

Inventors have utilized the zinc finger based transgene repair tool thatwas previously described by Tovkach et al. [Tovkach et al. (2009),supra] in order to generate petunia and tobacco transgenic plantscarrying a mutated uidA (GUS) gene. The mutated uidA gene was engineeredto carry the TGA (stop) codon within the 6-bp spacer of the QEQ-ZFNtarget site (see FIG. 25A), leading to premature termination of uidAtranslation in plant cells. Thus, no GUS expression was detectable inthe transgenic plants (FIG. 26I). Digestion of the DNA at the spacerbetween the ZFNs target site (by the use of specific ZFNs) and itssuccessive repair typically lead to deletion and/or mutation of the stopcodon and to the consequent activation of the uidA reporter gene (FIG.25B). To this end, QEQ-ZFN specific for the mutated uidA gene was clonedinto pTRV2-Δ2b-2sgP viral vector (as depicted in detail in Example 1,hereinabove) and the resultant pTRV2-Δ2b-sgP-QEQ-ZFN was used forinoculation of plants transgenic for mutated uidA gene. As clear fromthe results (FIGS. 26A-J), TRV-driven expression of the QEQ-ZFN insomatic and meristematic tissues lead to activation of GUS expression.

Example 12 Molecular Analysis of Transgenic Tobacco and Petunia Plantswith Activated GUS Expression Following Inoculation withpTRV2-Δ2b-sgP-QEQ-ZFN

Total plant DNA was extracted from leaves of GUS transgenic petunia andtobacco plants (carrying a mutated uidA (GUS) gene) before or 7-30 daysafter inoculation with pTRV1 and pTRV2-42b-sgP-QEQ-ZFN using thephenol-chloroform method. Total DNA was digested with DdeI for 3 hoursand the region surrounding the ZFN target site was PCR-amplified usingprimers 5′-CTATCCTTCGCAAGACCCTTCC-3′ (35S-F, SEQ ID NO: 90) and5′-GTCTGCCAGTTCAGTTCGTTGTTC-3′ (GUS-R-401, SEQ ID NO: 91). The resultingPCR fragment was redigested with DdeI, and its undigested fraction wasreamplified and TA cloned into pGEM-T-easy (Promega inc., WI, USA).Randomly selected colonies were then selected and the DNA fragmentssequenced.

FIG. 27 shows the changes in the GUS sequence following activation byQEQ-ZFN, as compared to the original GUS sequence in the transgenic N.tabacum or Petunia plants.

Example 13 Molecular Analysis of Modified PDS in Petunia PlantsFollowing Inoculation with Specific ZFNs

As depicted in detail in Example 1, hereinabove, inventors of thepresent invention have generated ZFNs which specifically cleave the PDSgene of petunia plants. To analyze the molecular modifications made tothe PDS gene following inoculation withpTRV2-42b-sgP-PDS-ZFN1-T2A-PDS-ZFN2, total plant DNA was extracted fromleaves of wild type (WT) or pTRV1 andpTRV2-42b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 treated Petunia hybrida plants usingthe phenol-chloroform method. The region surrounding the ZFN target site(TS) was PCR-amplified using primers 5′-TATTGAGTCAAAAGGTGGCCAAGTC-3′(phPDS-F 208, SEQ ID NO: 117) and 5′-GCAGATGATCATATGTGTTCTTCAG-3′(phPDS-R-487, SEQ ID NO: 118). The PCR product was digested with MfeIovernight and the resulting undigested fraction was reamplified and TAcloned into pGEM-T-easy (Promega inc., WI, USA). Inserts from randomlyselected colonies were then sequenced. FIG. 28B depicts the changes inthe PDS sequence following modification by the specific PDS-ZFNs, ascompared to the original PDS sequence in Petunia plants.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1. A method of generating genotypic variation in a genome of a plant,the method comprising introducing into the plant at least one viralexpression vector encoding at least one chimeric nuclease whichcomprises a DNA binding domain, a nuclease and a localization signal toa DNA-containing organelle, wherein said DNA binding domain mediatesspecific targeting of said nuclease to the genome of the plant, therebygenerating genotypic variation in the genome of the plant.
 2. A methodof treating a plant infection by a pathogen, the method comprisingintroducing into the plant at least one viral expression vector encodingat least one chimeric nuclease which comprises a DNA binding domain anda nuclease, wherein said DNA binding domain mediates targeting of saidnuclease to the genome of the pathogen, thereby preventing or treating aplant infection by a pathogen.
 3. (canceled)
 4. A method of generatingmale sterility in a plant, the method comprising upregulating in theplant a structural or functional gene of a mitochondria or chloroplastassociated with male sterility by introducing into the plant at leastone viral expression vector encoding at least one chimeric nucleasewhich comprises a DNA binding domain, a nuclease and a mitochondria orchloroplast localization signal and a nucleic acid expression constructwhich comprises at least one heterologous nucleic acid sequence whichcan upregulate said structural or functional gene of a mitochondria orchloroplast when targeted into the genome of said mitochondria orchloroplast, wherein said DNA binding domain mediates targeting of saidheterologous nucleic acid sequence to the genome of the mitochondria orchloroplast, thereby generating male sterility in the plant.
 5. A methodof generating a herbicide resistant plant, the method comprisingintroducing into the plant at least one viral expression vector encodingat least one chimeric nuclease which comprises a DNA binding domain, anuclease and a chloroplast localization signal, wherein said DNA bindingdomain mediates targeting of said nuclease to a gene conferringsensitivity to herbicides, thereby generating the herbicide resistantplant.
 6. A plant viral expression vector comprising a nucleic acidsequence encoding at least one chimeric nuclease which comprises a DNAbinding domain, a nuclease and a localization signal to a DNA-containingorganelle.
 7. A pTRV based expression vector comprising a nucleic acidsequence encoding at least two heterologous polypeptide sequences. 8.(canceled)
 9. A transgenic plant comprising the plant viral expressionvector of claim
 6. 10. (canceled)
 11. The method of claim 1, whereinsaid generating genotypic variation is transient. 12-13. (canceled) 14.The methods of claim 1, 2, 3, 4, 5 or 10, wherein said viral expressionvector comprises a Tobacco Rattle Virus (TRV) expression vector. 15.(canceled)
 16. The methods of claim 1, 2, 3, 4, 5 or 10, wherein said atleast one viral expression vector encodes for two chimeric nucleases.17-22. (canceled)
 23. The method of claim 1, wherein said at least onechimeric nuclease comprises two chimeric nucleases.
 24. The plant viralexpression vector of claim 6, further comprising a second nucleic acidsequence encoding a heterologous polypeptide.
 25. The plant viralexpression vector of claim 6, comprising a pTRV backbone. 26-27.(canceled)
 28. The plant viral expression vector claim 7, wherein saidnucleic acid sequence is devoid of a 2b sequence (SEQ ID NO: 43). 29.The plant viral expression vector claim 7, wherein said nucleic acidsequence comprises a Ω enhancer (SEQ ID NO: 44).
 30. The pTRV basedexpression vector of claim 7, wherein said nucleic acid sequencecomprises two separate sub genomic promoters (sgPs) for regulatingtranscription of said at least two heterologous polypeptides.
 31. ThepTRV based expression vector of claim 7, wherein said at least twoheterologous polypeptide sequences are separated by nucleic acidsequence encoding a cleavage domain.
 32. The pTRV based expressionvector of claim 31, wherein said cleavage domain comprises a T2A-likeprotein sequence (SEQ ID NO: 40).
 33. (canceled)
 34. The pTRV basedexpression vector or transgenic plant of claim 32, wherein said aminoacid sequence of at least two heterologous polypeptide sequences are asset forth in SEQ ID NOs: 85, 87 or
 89. 35. (canceled)
 36. The pTRV basedexpression vector of claim 7, wherein said at least two heterologouspolypeptide sequences comprise chimeric proteins, wherein each of saidchimeric proteins comprise a DNA binding domain, a nuclease and alocalization signal to a DNA-containing organelle. 37-38. (canceled) 39.The methods of claim 1, wherein said DNA binding domain binds a 9nucleotide sequence.
 40. The methods of claim 1, wherein said DNAbinding domain comprises three zinc finger domains. 41-43. (canceled)44. The methods of claim 1, wherein the plant comprises a Petuniahybrida or a Nicotiana tabacum. 45-48. (canceled)
 49. The methods ofclaim 44, wherein said specific targeting of said nuclease to the genomeof said Petunia hybrida is to a phytoene desaturase (PDS) or a flavanone3 beta-hydroxylase (FHT) of said Petunia hybrida. 50-52. (canceled) 53.An isolated polypeptide comprising an amino acid sequence as set forthin SEQ ID NOs: 35, 36, 37, 38, 71, 73, 75, 77, 85, 87 or 89.